Method for increasing viability and transformation efficiency of bacteria during storage at low temperatures

ABSTRACT

The invention relates to improved  E. coli  bacteria with enhanced viability at low temperatures, methods for producing improved bacterial strains capable of enhanced viability at low temperatures, and the isolation and use of genetic material capable of enhancing the viability of bacteria at low temperatures. In addition to the enhanced viability at low temperatures, the bacteria may exhibit enhanced transformation efficiencies after storage at low temperatures. As such, the invention may be used for the insertion of exogenous DNA sequences into the bacteria of the invention.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part of U.S. PatentApplication Serial No. 60/014,330, filed Mar. 29, 1996, hereinincorporated by reference and of U.S. Patent Application Serial No.60/025,838, filed Sep. 5, 1996, herein incorporated by reference.

FIELD OF THE INVENTION

[0002] The invention relates to stable storage of bacteria at lowtemperatures (e.g., about 4° C. to about −20° C. ). Specifically, theinvention relates to improved bacteria having enhanced viability orenhanced transformation efficiency during storage at low temperatures,methods for producing such bacteria, and the genetic material involvedin such enhancement. The invention further relates to such cells madecompetent for transformation, to methods for making such competantcells, and to methods of transforming such competent cells.

BACKGROUND OF THE INVENTION

[0003] Extended storage of bacteria is typically accomplished at verylow temperatures (−80° C. and below). Not only have such very lowtemperatures been used to store bacteria, but they have also been usedto store bacteria made competent for transformation (U.S. Pat. No.4,981,797). However, problems are associated with the storage ofbacteria and competent bacterial cells at higher temperatures (e.g.,about −20° C. to about 4° C.). At these higher temperatures, bacteriaand competent bacterial cells rapidly lose viability and transformationefficiency. Over a period of several months, the viable cell count andthe transformation efficiency of such cells decreases by several ordersof magnitude. Bacteria which are competent for transformation can onlybe stored at 4° C. for several days (Dagent et al., Gene 6:23-28 (1979))or for a period of up to 16 days (Pope et al., Nucl. Acids Res.24(3):536-537 (1996)). Thus, in order to maintain viability andcompetency, bacteria and competent bacterial cells have typically beenstored at −80° C.

[0004] Stable storage of bacteria, and particularly competent cells, attemperatures higher than −80° C. is highly desirable, since manyresearch laboratories may not have access to very low temperaturestorage. Moreover, the cost of storing bacteria at −80° C. is greaterthan at higher temperatures, and it is both difficult and expensive totransport bacterial cells at temperatures lower than −20° C.

SUMMARY OF THE INVENTION

[0005] The present invention provides a method which allows bacterialcells and competent cells to be stored for extended periods of time attemperatures greater than −80° C. (e.g., about −20° C. to about 4° C.)without appreciably losing transformation efficiency or viability. Thus,the method of the invention provides bacterial cells and competent cellswhich do not require specialized storage conditions (e.g., extremely lowtemperatures) to maintain the viability and/or transformation efficiencyof such cells.

[0006] The method of the invention specifically comprises altering thefatty acid content of the bacteria. Preferably, the unsaturated fattyacid content of the bacteria is altered in accordance with theinvention. Preferably, one or more of the fatty acids is increased inthe bacteria, and most preferably the fatty acid content is in thebacterial membrane. Preferred methods of altering the fatty acid contentincludes genetic alteration of the bacteria (e.g. by enhancingexpression of one or more genes involved in production (synthesis orcatabolism) of one or more fatty acids). Bacteria used according to theinvention include both gram positive and gram negative bacteria,although gram negative bacteria such as Escherichia are preferred.Particularly preferred bacteria include Escherichia coli.

[0007] The invention also relates to bacteria having enhanced viabilityand/or enhanced transformation efficiency after periods of storage atlow temperatures (e.g., greater than −80° C., preferably about −20° C.to about 4° C.). Such storage stable cells comprise an altered fattyacid content. Preferably, the storage stable bacterial cells orcompetent cells have an increased level or amount of one or more fattyacids, preferably unsaturated fatty acids. Such increased amount offatty acid content may be caused by genetic alterations, preferably byenhancing expression of one or more genes involved in changing the fattyacid content of the bacteria.

[0008] The invention also relates to methods of making the bacteria ofthe invention competent, to methods of transforming such competentbacterial strains of the invention, and to the bacterial cells of theinvention transformed with exogenous DNA. According to the invention,exogenous DNA sequences (e.g., plasmids, cosmids, DNA libraries, cDNAlibraries, expression vectors, eukaryotic (particularly mammalian, andmost particularly human) DNA, phage DNA, etc.) may be transformed intothe novel bacteria of the invention by any of a variety of techniques,including, but not limited to, chemical-mediated transformation,electroporation, and liposome-mediated transformation.

[0009] The invention further provides a DNA molecule comprising asequence capable of enhancing the viability or the transformationefficiency of a bacterium at low temperatures (e.g., greater than −80°C., preferably about −20° C. to about 4° C.). Such DNA moleculepreferably comprises one or more genes involved in the production of oneor more fatty acids in the bacteria. Most preferably, the nucleic acidmolecule of the invention allows enhanced production of fatty acids inthe bacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 shows the viability of E. coli DH10B, SB3499A, SB3499B, andSB3499C expressed in terms of cells/ml and after storage of the cells at20° C.

[0011]FIG. 2 shows the transformation efficiency of E. coli DH10B,SB3499A, SB3499B, and SB3499C expressed in terms of transformants per RgDNA after storage of the cells at −20° C.

[0012]FIG. 3 shows the viable cell counts of DH10B bacteria containingeither the cosmid vector pCP13, or cosmid 1 (NRRL B-21550), 2 (NRRLB-21551) or 4 (NRRL B-21552) after those cells are stored at 4° C. forperiods of time.

[0013]FIG. 4A shows the viable cell counts of DH10B cells containingvector pCP13 and cosmids 1, 2 and 4 over a period of 4 months duringwhich the cells are stored at −20° C.

[0014]FIG. 4B shows the viable cell counts of DH10B cells cured ofclones 1, 2, and 4 after these cells are stored at 4° C. for periods oftime.

[0015]FIG. 5 shows the viable cell counts of DH10B cells, DH10B cellscontaining vector pCP13 or cosmid 1, DH10B cured of cosmid 1, DH10Bcured of cosmid 1 and retransformed with vector pCP13, or cosmid 1 andDH10B cells containing cosmid 1 after those cells are stored at 4° C.for periods of time.

[0016]FIGS. 6A and 6B show the viability of the following bacterialstrains stored at 4° C.: DH5α, DH5α containing the vector pCP13, DH5αcontaining cosmid 1, STBL2, STBL2 containing the vector pCP13, and STBL2containing cosmid 1 after those cells are stored at −20° C. for periodsof time.

[0017]FIG. 7 shows the transformation efficiencies of the DH10B straincontaining cosmid clone 1, and the DH10B strain containing vector pCP13,both stored at −20° C. for 5 months.

[0018]FIG. 8 shows the viability of DH10B cells containing either cosmidclone 1, pDELTA2, pDELTA2 8Kb+, pDELTA2 8Kb−, pDELTA2 14Kb+, or pDELTA214Kb− plasmid after storage at 4° C.

[0019]FIG. 9 shows the viability of DH10B cells containing either cosmidclone 1, pDELTA2, pDELTA2 8Kb+, pDELTA2 8Kb−, pDELTA2 14Kb+, or pDELTA214Kb− plasmid after storage at −20° C.

[0020]FIG. 10 shows the viability of DH10B cells containing either thepDELTA2, pDELTA2 14Kb+, pDELTA2 14Kb− plasmid, or one of eleven deletionderivatives of the pDELTA2 14Kb+plasmid generated by the DeleletionFactory System under sucrose/ampicillin selection after storage at 4° C.for various intervals.

[0021]FIG. 11 shows the viability of DH10B cells containing either thepDELTA2, pDELTA2 14Kb+, plasmid, or one of sixteen deletion derivativesof the pDELTA2 14Kb+generated by the Deleletion Factory System understreptomycin/kanamycin selection after storage at 4° C. for variousintervals.

[0022]FIG. 12 combines the stability studies outlined in FIGS. 10 and 11and presents the length of the insert DNA remaining after deletion of aportion of the insert using the Deletion Factory System.

[0023]FIG. 13 shows DNA sequence (SEQ ID NO: 14) of the essential regionof cosmid clone 1 (2658 bp).

[0024]FIG. 14 depicts the open reading frames and location of MluIrestriction sites contained within the essential region of cosmid clone1.

[0025]FIG. 15 shows the viability of DH10B cells containing either thepDELTA2, pDELTA2 32, pDELTA2 fabB10, pDELTA2 fabB13, pDELTA2 fabB14, orpDELTA2fabB15 plasmid after storage at 4° C. for 9 days.

[0026]FIG. 16 shows the viability of DH10B cells containing either thepDELTA2, pDELTA2 32, pDELTA2 fabB10, pDELTA2 fabB13, pDELTA2 fabB14, orpDELTA2fabB15 after storage at −20° C. (for approximately 120 days).

[0027]FIG. 17 shows the viability of DH10B cells containing either thepDELTA2, pDELTA2 fabB14, pDELTA2 14 deletion, pDELTA2 fabB15 or pDELTA215 deletion plasmid after storage at 4° C. for up to 30 days.

[0028]FIG. 18 shows the viability of DH10B cells containing either thepDELTA2, pDELTA2 fabB14, pDELTA2 14 deletion, pDELTA2 fabB15 or pDELTA215 deletion plasmid after storage at −20° C. for up to 60 days.

[0029]FIG. 19 shows the effect of the presence of various fabB clones instrains DH5α and DH10B on percent cis vaccenic acid levels.

[0030]FIG. 20 shows the effect of the presence of various fabB clones instrains DH5α and DH10B on percent unsaturated fatty acids levels.

[0031]FIG. 21 shows the viability of DH5α cells containing either thepCP13, cosmid clone 1, pDELTA2, or pDELTA2fabB15 plasmid after storageat −20° C. for up to 2 months.

[0032]FIG. 22 shows the viability of DH10B cells containing either thepCP13, cosmid clone 1, pDELTA2, or pDELTA2 fabB15 plasmid after storageat −20° C. for up to 2 months.

[0033]FIG. 23 shows the correlation that exists between the survival ofDH5α and DH10B cells at −20° C. for two months and the amount ofunsaturated fatty acids found in the cell membrane.

[0034]FIG. 24 shows the correlation that exists between the growthtemperature of DH10B, SB3499B, and CY322 cells grown at 23° C., 30° C.,37° C., or 42° C. for 16 hours, and the amount of cis vaccenate in thecells.

[0035]FIG. 25 shows the effect of varying growth temperature on thelevels of unsaturated fatty acids in the cell membrane of DH10B,SB3499B, and CY322 cells grown at 23° C., 30° C., 37° C., or 42° C.

[0036]FIG. 26 shows the viability of DH10B cells grown at 23° C., 30°C., 37° C., or 42° C. after storage at −20° C. for up to 2 months.

[0037]FIG. 27 shows the viability of SB3499B cells grown at 23° C., 30°C., 37° C., or 42° C. after storage at −20° C. for up to 2 months.

[0038]FIG. 28 shows the corelation between the total unsaturated fattyacids of the cell membrane and cell survival at −20° C. for strainsDH10B and SB3499B.

DETAILED DESCRIPTION OF THE INVENTION:

[0039] Definitions

[0040] In the description that follows, a number of terms used inrecombinant DNA technology are utilized extensively. In order to providea clear and consistent understanding of the specification and claims,including the scope to be given such terms, the following definitionsare provided.

[0041] DNA Molecule.

[0042] Any DNA molecule, of any size, from any source, including DNAfrom viral, prokaryotic, and eukaryotic organisms. The DNA molecule maybe in any form, including, but not limited to, linear or circular, andsingle or double stranded. Non-limiting examples of DNA moleculesinclude plasmids, vectors, and expression vectors.

[0043] Cloning Vector.

[0044] A plasmid, phage DNA, a cosmid, or other DNA molecule which isable to replicate autonomously in a host cell, and which ischaracterized by one or a small number of restriction endonucleaserecognition sites at which such DNA sequences may be cut in adeterminable fashion without loss of an essential biological function ofthe vector, and into which a DNA fragment may be spliced in order tobring about its replication and cloning. The cloning vector may furthercontain a marker suitable for use in the identification of cellstransformed with the cloning vector. Markers, for example, providetetracycline resistance or ampicillin resistance.

[0045] Expression Vector.

[0046] A vector similar to a cloning vector but which is capable ofexpressing a gene which has been cloned into it, after transformationinto a host. The cloned gene is usually placed under the control of(i.e., operably linked to) certain control sequences such as promotersequences.

[0047] Storage Stable.

[0048] Within the meaning of the present invention, bacterial cellsand/or competent bacterial cells which are “storage stable” are able towithstand storage for extended periods of time at a suitabletemperature, without appreciably losing their transformation efficiencyand/or viability. By the term “without appreciably losing theirtransformation efficiency and/or viability” is meant that the cellsmaintain about 40% to 100%, preferably 60% to 100%, more preferably 70%to 100%, and most preferably about 80% to 100% of their originaltransformation efficiency and/or viability during a storage period of 30days, preferably 60 days, more preferably 90 days, and most preferably120 days, at a temperature of −20° C. Suitable storage temperatures forthe bacterial cells or competent bacterial cells of the invention varyfrom about room temperature to about −180° C. Preferably, the storagetemperature ranges from about 4° C. to about −80° C., more preferablyfrom about −20° C. to about 4° C. In a preferred aspect of theinvention, the cells are stored at about −20° C. The storage period ortime may range from about 0 days to about 180 days (e.g., 6 months),preferably from about 0 days to about 120 days (e.g., 4 months), andmore preferably from about 0 days to about 90 days (e.g., 3 months),although longer storage times may be used at temperatures of about −20°C. and below.

[0049] Fatty Acids.

[0050] Any fatty acid which is saturated or unsaturated. Unsaturatedfatty acids including monoenic acids, dienoic acids and higherunsaturated fatty acids (e.g., tri, tetra, penta and hexaenoic acidsetc.). Examples of unsaturated fatty acids include but are not limitedto oleic acid, linoleic acid, linolenic acid, cis vaccenic acid,arachidonic acid, palmitoleic acid etc. Examples of saturated fattyacids include butyric acid, lauric acid, palmitic acid and stearic acid.Preferred fatty acids in accordance with the invention are unsaturatedfatty acid, most preferably cis vaccenic acid and palmitoleic acid.

[0051] Competent Cells.

[0052] Cells having the ability to take up and establish an exogenousDNA molecule.

[0053] The present invention relates to a method for enhancing viabilityor transformation efficiency of a bacterium by altering the fatty acidcontent (preferably the unsaturated fatty acid content). The inventionalso relates to a method for obtaining a bacterium having such analtered fatty acid content. The method involves modifying or mutating abacterium such that the fatty acid content of said bacterium is alteredrelative to an unmodified or unmutated bacterium. The modified ormutated bacterium having enhanced viability or enhanced transformationefficiency may then be isolated. Selection of such a modified bacteriummay be selected by assaying for such enhanced characteristics relativeto the unmodified bacterium (see Examples). Preferably, the amount offatty acid is increased in the bacterium. This increase may beaccomplished by various techniques, for example, by adding one or morefatty acids to the bacterium or by genetically modifying the bacterium.Any type of genetic modification may be used in accordance with theinvention, including natural selection, artificial mutation and geneticengineering. Such techniques are well known in the art. Common geneticengineering techniques include cloning one or more fatty acid genes in avector to increase copy number, or enhancing translation ortranscription of such genes by, for example, overexpression (e.g., usingan expression vector).

[0054] The method of the invention provides for the production of cellswhich have enhanced viability and/or enhanced transformation efficiencyupon storage at low temperatures (e.g., greater than −80° C., preferablyabout −20° C. to about 4° C.). Such storage stable strains may be storedfor extended periods at various temperatures. According to theinvention, alteration of the content of one or more fatty acids resultsin enhanced viability or enhanced transformation efficiency. Alterationof the fatty acid content can be accomplished in any bacteria to providebacteria having these beneficial characteristics. Preferably, suchbacteria are modified genetically.

[0055] Both gram negative and gram positive prokaryotic cells (e.g.,bacteria) can be used in accordance with the invention. Examples ofsuitable prokaryotic cells include, but are not limited to, Escherichiasp., Klebsiella sp., Salmonella sp., Bacillus sp., Streptomyces sp.,Streptococcus sp., Shigella sp., Staphylococcus sp., and Pseudomonas sp.Non-limiting examples of species within each aforementioned genus thatcan be used in accordance with the invention include Escherichia coli,Klebsiella pneumoniae, Bacillus subtilis, Salmonella typhimuritum,Streptomyces aureus, Streptococcus mutans, Streptococcus pneumoniae, andPseudomonas syringae.

[0056] In a preferred embodiment, the cells which can be used in theinvention are Escherichia, most preferably E. coli. Non-limitingexamples of E. coli strains include DH5, DH5α, DH10, DH10B, HB101, RR1,JV30, DH11S, DM1, DH10B/p3, DH5αMCR, DH5αF′IQ, DH5αF′, SCS1, Stbl-2,DH12S, DH5α-E, DH10BAC, XL1-Blue MRF, XL2-Blue MRF, XL1-Blue MR, SUREStrain, SURE 2 Strain, XL1-Blue, XL2-Blue, AG1, JM101, JM109,JM110/SCS110, NM522, TOPP Strains, ABLE Strains, XL1-Red, BL21 Strains,BJS183 TK B1 Strain, and derivatives thereof.

[0057] As used herein, a “derivative” of a specified bacterium is aprogeny or other recipient bacterium that contains genetic materialobtained directly or indirectly from the specified bacterium. Suchderivative bacterium may, for example, be formed by removing geneticmaterial from a specified bacterium and subsequently introducing it intoanother bacterium (i.e., the progeny or other recipient bacterium)(e.g., via transformation, conjugation, electroporation transduction,etc.). Alternatively, such derivative bacterium may possess geneticmaterial (produced synthetically, via cloning, via in vitroamplification, etc.) having the effective sequence of genetic materialof the specified bacterium.

[0058] The bacteria of the invention having altered fatty acid contentmay be made competent for transformation using well known techniques.Such competent bacterial cells have, according to the invention,enhanced transformation efficiency upon or after storage at lowtemperatures (e.g., greater than −80° C., preferably about −20° C. toabout 4° C.). Transformation, in the context of the current invention,is the process by which exogenous DNA is inserted into a bacterium,causing the bacterium to change its genotype and/or phenotype. Such achange in genotype or phenotype may be transient or otherwise. ExogenousDNA is any DNA, whether naturally occurring or otherwise, from anysource that is capable of being inserted into any organism. Preferably,exogenous DNA is any DNA, whether naturally occurring or otherwise, fromany source that is capable of being inserted into bacteria. Suchexogenous DNA includes, without limitation, plasmid DNA, cosmid DNA,eukaryotic (particularly mammalian, and most particularly human) DNA,DNA libraries, cDNA libraries, expression vectors and phage DNA (such asbacteriophage lambda DNA).

[0059] A number of procedures exist for the preparation of competentbacteria and the introduction of DNA into those bacteria. A very simple,moderately efficient transformation procedure for use with E. coliinvolves re-suspending log-phase bacteria in ice-cold 50 mM calciumchloride at about 10¹⁰ bacteria/ml and keeping them ice-cold for about30 min. Plasmid DNA (0.1 mg) is then added to a small aliquot (0.2 ml)of these now competent bacteria, and the incubation on ice continued fora further 30 minutes, followed by a heat shock of 2 minutes at 42° C.The bacteria are then usually transferred to nutrient medium andincubated for some time (30 minutes to 1 hour) to allow phenotypicproperties conferred by the plasmid to be expressed, e.g., antibioticresistance commonly used as a selectable marker for plasmid-containingcells. Protocols for the production of competent bacteria have beendescribed (Hanahan (J. Mol. Biol. 166: 557-580 (1983); Liu et al., BioTechniques 8:21-25 (1990); Kushner, In: Genetic Engineering: Proceedingsof the International Symposium on Genetic Engineering, Elsevier,Amsterdam, pp. 17-23 (1978); Norgard et al., Gene 3:279-292 (1978);Jessee et al., U.S. Pat. No. 4,981,797); Maniatis et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1982).

[0060] Another rapid and simple method for introducing genetic materialinto bacteria is electoporation (Potter, Anal. Biochem. 174: 361-73(1988)). This technique is based upon the original observation byZimmerman et al., J. Membr. Biol. 67: 165-82 (1983), that high-voltageelectric pulses can induce cell plasma membranes to fuse. Subsequently,it was found that when subjected to electric shock (typically a briefexposure to a voltage gradient of 4000-16000 V/cm), the bacteria take upexogenous DNA from the suspending solution, apparently through holesmomentarily created in the plasma membrane. A proportion of thesebacteria become stably transformed and can be selected if a suitablemarker gene is carried on the transforming DNA (Newman et al., Mol. Gen.Genetics 197: 195-204 (1982)). With E. coli, electoporation has beenfound to give plasmid transformation efficiencies of 10⁹-10¹⁰ T/μg DNA(Dower et al., Nucleic Acids Res. 16: 6127-6145 (1988)).

[0061] Bacterial cells are also susceptible to transformation byliposomes (Old and Primrose, In: Principles of Gene Manipulation: AnIntroduction to Gene Manipulation, Blackwell Science (1995)). A simpletransformation system has been developed which makes use of liposomesprepared from cationic lipid (Old and Primrose, In: Principles of GeneManipulation: An Introduction to Gene Manipulation, Blackwell Science(1995)). Small unilamellar (single bilayer) vesicles are produced. DNAin solution spontaneously and efficiently complexes with these liposomes(in contrast to previously employed liposome encapsidation proceduresinvolving non-ionic lipids). The positively-charged liposomes not onlycomplex with DNA, but also bind to bacteria and are efficient intransforming them, probably by fusion with the cells. The use ofliposomes as a transformation or transfection system is calledlipofection.

[0062] The present invention also concerns genetic material capable ofenhancing the viability and/or transformation efficiency of saidbacterium at low temperatures. In particular, the invention concernsisolated nucleic acid molecules which allow alteration of fatty acidcontent when introduced into a bacterium. Preferably, the nucleic acidmolecule comprises one or more genes involved in changing fatty acidcontent of said bacterium. Such genetic material is preferably containedin a cloning or expression vector. In one aspect of the invention, thenucleic acid molecule comprises one or more genes which enhance thelevel of one or more fatty acids. Preferably, the genes enhanceunsaturated fatty acid levels. Such unsaturated fatty acids include, butare not limited to, oleic acid, linoleic acid, linolenic acid, cisvaccenic acid, and palmitoleic acid. Such genes include but are notlimited to fabB, fabF, fabD, fabG, fabA, fabH, fabI, fabZ, fadA, fadB,fadE, fadL, fadR, farR, fatA, etc.

[0063] The viable cell count of cells produced by the method of theinvention will remain at greater than about 1×10⁶ cells/ml, preferablygreater than about 1×10⁷ cells/ml, more preferably greater than about1×10⁸ cells/ml and most preferably greater than 1×10⁹ cells/ml whenstored at −20° C. for any time period from about 0 days to about 1month, preferably from about 0 days to about 3 months, and morepreferably from about 0 days to about 6 months. These cells will retaina transformation efficiency of at least about 1×10⁵, preferably at leastabout 1×10⁶, more preferably at least about 1×10⁷ still more preferablyat least about 1×10⁸ and most preferably at least about 1×10⁹transformants per microgram of DNA (T/μg). Suitable storage temperaturesvary from about room temperature to about −180° C. Preferably, thestorage temperature ranges from about 4° C. to about −80° C., morepreferably from about −20° C. to about 4° C. In a preferred aspect ofthe invention, the cells are stored at about −20° C. The storage periodor time may range from about 0 days to about 1 month, preferably fromabout 0 days to about 3 months, still more preferably from about 0 daysto about 6 months, and still more preferably from about 0 days to about1 year, although longer storage times may be used at temperatures ofabout −20° C. and below. Competent cells produced by the method of theinvention may be stored at −20° C. for at least 3 months while retainingsubstantially their transformation efficiency. Substantial retention oftransformation efficiency means that the cells will have atransformation efficiency after storage that is about 40% to 100%,preferably at about 60% to 100%, more preferably about 70% to 100% andmost preferably about 80% to 100% of the transformation efficiency ofthe competent cells tested prior to storage.

[0064] The invention also pertains to transforming the competentbacterial cell produced according to the method of invention.Transforming said competent cells comprises obtaining a competentbacterial cell of the invention, mixing said cell with a DNA molecule,and incubating said mixture under conditions sufficient to transformsaid cell with said DNA molecule. According to this aspect of theinvention, the competent cell may be any gram positive or gram negativebacteria including, but not limited to, Escherichia, Klebsiella,Salmonella, Bacillus, Streptomyces, Streptococcus, and Pseudomonas.Preferably, gram negative prokaryotic cells are transformed according tothe method of the invention, more preferably Escherichia, and mostpreferably E. coli. According to the invention, any DNA molecule (e.g.,vectors, plasmids, phagemids, expression vectors, etc.) may be used.

[0065] After the cells have been transformed with the DNA molecule ofinterest, the transformed cells may be grown in a growth conducivemedium. Typically, such a growth conducive medium contains an antibioticto assist in selection of transformed cells. That is, the DNA moleculeto be transformed may contain a selective marker (e.g., an antibioticresistance gene), allowing selection of transformed cells when thecorresponding antibiotic is used in the medium.

[0066] The invention also concerns a method of producing a desiredprotein by transforming a competent cell of the invention with a DNAmolecule encoding said desired protein. Thus, the invention concerns amethod of producing a desired protein comprising obtaining a competentcell produced according to the invention, transforming said cell with aDNA molecule capable of expressing said desired protein, and culturingsaid transformed cell under conditions sufficient to produce saiddesired protein. Cells which can be used according to this aspect of theinvention including both gram negative and gram positive bacteria,preferably Escherichia, and most preferably E. coli. In this aspect ofthe invention, the cells are transformed by mixing the cells with a DNAmolecule and incubating the mixture under conditions sufficient totransform said cell with said DNA molecule. Transformed cells may beselected according to techniques well known in the art including, forexample, selection for marker genes on the DNA molecule (e.g.,antibiotic resistance genes). After the transformed cell has beenselected, the cell may then be cultured according to well knowntechniques in a growth conducive medium. Upon culturing the cell underappropriate conditions, the cell is capable of producing the desiredprotein. The desired protein may then be isolated, and a substantiallypure protein obtained by well known protein purification techniques.

[0067] Having now generally described the invention, the same will bemore readily understood through reference to the following exampleswhich are provided by way of illustration, and are not intended to belimiting of the present invention unless specified. All patents, patentapplications and publications mentioned herein are incorporated byreference in their entirety.

EXAMPLE 1 Generation of Mutant Strains by Cycling

[0068] A common technique in microbial genetics is the generation ofmutant strains of bacteria utilizing recycling. This technique isdependent on a rapid rate of bacterial killing under some unfavorablecondition. In this particular case, the storage of E. coli strain DH10Bin CCMB80 buffer at −20° C. results in the rapid killing of the strainand the subsequent decrease in the viable cell count over a period of 6months. The survivors which remain after 6 months storage at −20° C. areagain regrown and stored at −20° C. in CCMB80 buffer. After 4 cyclesunder these particular conditions single colonies are tested forsurvival at −20° C. The isolated strain was designated SB3499.

EXAMPLE 2 Preparation of Competent Cells

[0069] A single colony isolate of E. coli strain DH10B and three singlecolony isolates of E. coli SB3499 (Example 1) (designated SB3499A (NRRLB-21606), SB3499B (NRRL B-21607), and SB3499C (NRRL B-21608)respectively, each deposited on Aug. 1, 1996, under the terms of theBudapest Treaty governing Microbiological Deposits) were selected.Competent cells of these isolates were made according to the method ofJessee, J. and Bloom, F. R., U.S. Pat. No. 4,981,797, hereinincorporated by reference. Essentially the process is as follows: Thesingle colony isolates of DH10B, SB3499A, SB3499B, and SB3499C were eachinoculated into 2 ml of 15/10 medium (1.0% Bacto tryptone, 1.5% Bactoyeast extract, 10 mM NaCl, 2 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 0.001 %polypropylene glycol (PPG)) and shaken overnight at 275 rpm at atemperature of 30° C. A 0.3 m 1 aliquot of each overnight culture wasused to inoculate a 500 ml baffled flask containing 60 ml 15/10 medium.The resulting cultures were grown by shaking them at 275 rpm at atemperature of 30° C. until the O.D.₅₅₀ of the cultures wasapproximately 0.3. A 50 ml aliquot of the culture was harvested bycentrifugation of that aliquot at 2,000 rpm and 4° C., for a sufficienttime to pellet the bacterial cells. The bacterial cell pellets were thenresuspended in 4 ml of ice cold CCMB80 buffer (10 mM potassium acetatepH 7.0, 80 mM CaCl₂, 20 mM MnCl₂, 10 mM MgCl₂, 10% glycerol adjusted topH 6.4 with 0.1 N HCl, as described in Hanahan, et al., Methods inEnzymology, 204:63-113 (1991), herein incorporated by reference. Theresuspended bacterial cells were then kept on ice for 20 minutes. Theresuspended bacterial cells were then divided into 250 μl aliquots andfrozen in a dry ice/ethanol bath for at least 5 minutes. All but onealiquot of each isolate was stored at −20° C. for a specified period oftime prior to a determination of its viability and its transformationefficiency.

EXAMPLE 3 Viability and Transformation Assays

[0070] Aliquots containing competent cells prepared according to Example2 were tested after a specified period of storage at −20° C. todetermine the viability and transformation efficiency of that aliquot.Aliquots were either tested immediately or at monthly intervals afterstorage at −20° C.

[0071] To determine the viability of an aliquot, the aliquot was removedfrom −20° C. and placed on ice for approximately 15 minutes. The aliquotwas serially diluted using 0.85% NaCl. Dilutions were then plated on LBagar plates (Gibco/BRL) to determine viable cell counts. FIG. 1 showsthe viability of E. coli DH10B, SB3499A, SB3499B, and SB3499C. Theviable cell counts of SB3499A, SB3499B and SB3499C were over 100 foldhigher than DH10B after these cells were stored for between 2-4 monthsat −20° C.

[0072] To determine the transformation efficiency of an aliquot, thealiquot was removed from −20° C. and placed on ice for approximately 15minutes. The cells were assayed for transformation efficiency usingplasmid pUC19, according to the method of Hanahan, J. Mol. Biol. 166:577 (1983), herein incorporated by reference. FIG. 2 shows thetransformation efficiency of E coli DH10B, SB3499A, SB3499B, and SB3499Cexpressed in terms of transformants per jig DNA. The transformationefficiency of DH10B cells decreased to less than 1.0×10⁵transformants/,g DNA after one month of storage at −20° C. Thetransformation efficiencies of SB3499A, SB3499B, and SB3499C weregreater than 3.0×10⁶ transformants/μg DNA after four months of storageat −20° C. Therefore isolates SB3499A, SB3499B and SB3499C exhibitsubstantially higher viable cell counts and transformation efficienciesthan DH10B after prolonged storage at −20° C.

EXAMPLE 4 Genetic Characterization of E. coli DH10B, SB3499A, SB3499B,and SB3499C

[0073] The SB3499A, SB3499B and SB3499C isolates were evaluated forgenetic markers that are characteristic of E. coli DH10B. Table 1 showsthe characteristic genetic markers for the SB3499A, SB3 499B, andSB3499C isolates of the E. coli strain DH10B. The genetic markers whichwere evaluated for strains SB3499A, SB3499B and SB3499C are identical tothe genetic markers of DH10B. MacConkey galactose plates are made usingDifco MacConkey agar containing 1% galactose. X-gal IPTG plates weremade using LB agar containing 50 μg/ml X-gal and 1 mM IPTG. Minimal agarplates were made by adding 100 ml of stock I (20 g ammonium chloride, 60g potassium phosphate (monobasic), and 120 g sodium phosphate (dibasic),diluted to 1 liter with distilled water), 60 ml of stock II (60 gglucose, and 1.3 g magnesium sulfate heptahydrate diluted to 600 ml withdistilled water), 20 ml of stock mH (0.735 g calcium chloride dehydratediluted to 200 ml with distilled water) and 1 ml 0.1% thiamine to 12g ofBacto agar and adjusting the final volume to 600 ml. For platescontaining leucine, 100 μl of a 1.5% solution of leucine was spread onthe plates. TABLE 1 Analysis of Genetic Markers Culture Bacterial StrainMedium DH10B SB3499A SB3499B SB3499C Minimal No Growth No Growth NoGrowth No Growth Minimal + Growth Growth Growth Growth Leucine MacConkeyWhite White White White Galactose Colonies Colonies Colonies ColoniesLB + No Growth No Growth No Growth No Growth Nitrofurantoin 8 μg/ml LB +No Growth No Growth No Growth No Growth Ampicillin 100 μg/ml LB + WhiteWhite White White X gal-IPTG Colonies Colonies Colonies Colonies LB +Naladixic No Growth No Growth No Growth No Growth Acid LB + No Growth NoGrowth No Growth No Growth Kanamycin 50 μg/ml LB + No Growth No GrowthNo Growth No Growth Chlor- amphenicol 10 μg/ml LB + No Growth No GrowthNo Growth No Growth Tetracycline 15 μg/ml

EXAMPLE 5 Genomic DNA Isolation

[0074] Genomic DNA isolation is described by Lin and Kuo, Focus 17:66-70 (1995), herein incorporated by reference. E. coli strain BRL 3433(Life Technologies, Inc.) was streaked on LB plates and incubatedovernight at 37° C. A single bacterial colony was isolated andresuspended in 5 ml LB broth (10 grams tryptone, 5 grams yeast extract,5 grams NaCl per liter) and then further incubated at 37° C./275 rpm ina shaking incubator overnight. A 1.0 ml aliquot of the cultured cellswas transferred into a microcentrifuge tube and the cell pellets arecollected by centrifuging at 11,000× g for 5 minutes at roomtemperature. The pellets were resuspended in 1 ml TES-sucrose buffer [8%sucrose, 50 mM NaCl, 20 mM Tris-HCl (pH 8.0), and 1 mM EDTA] andincubated at 25° C. for 5 minutes with 1 mg/ml lysozyme. A volume of 100μl of 10% SDS was added to the tube and the tube was briefly vortexed.The DNA was extracted with phenol-chloroform and precipitated with 0.3 Msodium acetate and 1 volume of isopropanol. The solution was centrifugedat 11,000× g for 10 minutes at 4° C. The pellet was washed with 70%ethanol and centrifuged at 11,000× g for 10 minutes at 4° C. Toeliminate RNA contamination, the DNA pellet was resuspended with 200 μlTE buffer and then treated with 1 μg/ml RNase A at 37° C. for 10minutes. The sample was then extracted with phenol-chloroform and theDNA was precipitated with 0.3 M sodium acetate and 2.5 volumes of 100%ethanol. The solution was then centrifuged at 11,000× g for 15 minutesat 4° C. The DNA pellet was washed with 70% ethanol and centrifuged at11,000× g for 10 minutes at room temperature. After washing the DNApellet, it was dissolved in 100 μl TE buffer.

EXAMPLE 6 Construction of Cosmid Genomic Library of E. coli Strain

[0075] 100 μg DNA of genomic DNA isolated from BRL 3433 (Example 5) wasmixed with 10× React 2 (Life Technologies, Inc.) and sterilizeddistilled water in order to obtain a final concentration of 100 μg/mlDNA in 1× React 2 buffer and in a final reaction volume of 1000 μl. Thegenomic DNA sample was aliquoted into 9 microtubes with the first tubecontaining 200 μl of genomic DNA and the other 8 tubes containing 100 μlof the genomic DNA. A 5 μl aliquot was removed from the last tube (tube9) and was saved as an untreated control.

[0076] 10 units of the restriction endonuclease PstI (Life Technologies,Inc.) was added to tube one. A two fold serial dilution was set up asfollows: 100 μl of the sample (including enzyme) was transferred fromtube one to the second tube, and so on, in order to obtain a series of 2fold dilutions of PstI concentrations-in tubes containing equalconcentrations of genomic DNA (10 μg DNA per tube). Finally, 100 μl fromthe tube 9 was transferred into an additional tube (numbered 10). All 10tubes were incubated at 37° C. for exactly 1 hour, and immediatelystored at −20° C. until use.

[0077] All ten tubes were thawed on ice and a 3 μl aliquot from eachtube in the serial dilution series was compared to the 5 μl un-cutcontrol by agarose gel electrophoresis (0.9% agarose gel) in order toelucidate the extent to which the genomic DNA was digested. The serialdilution sample or samples, which contained the highest percentage ofDNA fragments in the range of 25 to 45 kb estimated by agarose gelelectrophoresis according to the method set out in Maniatis et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (1982), was chosen to construct the cosmid library.These chosen aliquot or aliquots were extracted twice with phenol andthen ethanol precipitated with 2.5 volumes of 1000% ethanol andcentrifuged at 11,000 g for 30 minutes. The DNA pellet was washed with200 μl 70% ethanol and centrifuged at 11,000× g for 10 minutes. Theresulting partially digested genomic DNA pellet (termed “genomic digestDNA”) was resuspended in 15 μl 1× T4 DNA ligase buffer (LifeTechnologies, Inc.).

[0078] 5 μg of BJS80 (pCP13) cosmid vector was incubated for 4 hours at37° C. with 30 units of PstI (Life Technologies, Inc.) in 1× React 2(Life Technologies, Inc.) in a final volume of 50 μl. The reaction mixcontaining the cosmid vector was then ethanol precipitated with 2.5volumes of 100% ethanol and centrifuged at 11,000× g for 30 minutes. Theresulting DNA pellet was washed with 200 μl 70% ethanol and thencentrifuged at 11,000× g for 10 minutes. The washed pellet was finallyresuspended in 50 Al of lx React 2 buffer (Life Technologies, Inc.). A 1μl aliquot of calf intestinal alkaline phosphatase ((1 unit/μl) (LifeTechnologies, Inc.)) was mixed with the pre-cut cosmid vector andincubated at 37° C. for more than 30 minutes to remove the 5′ phosphate.A 5 λl aliquot of the sample was electrophoresed on a 0.9% agarose gelto confirm that the sample was linear according to the method ofManiatis et al., Molecular Cloning, a Laboratory Manual, Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (1982). The remainder of thesample was then phenol extracted twice and ethanol precipitated with 2.5volumes of 100% ethanol and centrifuged at 11,000× g for 30 minutes. Theresulting DNA pellet was washed with 200 μl of 70% ethanol and thencentrifuged at 11,000× g for 10 minutes. The washed pCP13 cosmid vectorwas then resuspended in 1,5 μl 1× T4 DNA ligase buffer (LifeTechnologies, Inc.) and stored at −20° C. until needed.

[0079] The stored cosmid vector was thawed on ice. 15 μl of thawedvector (100 ng/μl) was added to 15 μl of PstI digested “genomic digestDNA” (600 ng/ml) and 1 μl of 1 unit/μl T4 DNA ligase (“ligation reactionmixture”) (Life Technologies, Inc.)). The “ligation reaction mixture”was incubated overnight at room temperature and stored at −20° C. untilneeded.

[0080] The ligated DNA was packaged by utilizing the λ packaging kit(the MaxPlax Packaging Extract) from Epicentre Technologies, 1207 AnnStreet, Madison, Wis. 53713. The packaging procedure was as described inthe manufacture's instructions, herein incorporated by reference. Thereaction was essentially as follows: from the MaxPlax kit, one tube offreeze thawed sonicate extract, stored at −70° C., was thawed at roomtemperature. To the thawed extract was immediately added 5 μl of the“ligation reaction mixture” (about 2 μg template DNA). After the samplewas centrifuged at 11,000× g for 2 seconds it was incubated at 22° C.for 2 hours. After incubation, 500 μl of phage buffer (10 mM Tris-HCl,100 mM NaCl, 10 mM MgCl₂, and NaOH to pH 8.3) was added to the sample. A200 μl aliquot was removed from the sample and used to infect DH10Bcells ((Life Technologies, Inc.) (Hanahan et al., Methods in Enzymology,204: 63-111 (1991), herein incorporated by reference)) as describedbelow (“retained phage stock”). The remainder of the sample was mixedwith 15 μl chloroform and stored at 4° C. as a phage stock for futureuse.

[0081] From an overnight culture, a 200 μl aliquot of a culture of DH10Bwas used to inoculate 25 ml of LB medium. The cells were grown at 37° C.shaking at 275 rpm. When the cell density reached O.D.₅₉₀, 0.5, thecells were centrifuged at 11,000 g for 2 minutes and then resuspended in2 ml of 10 mM MgSO₄. 200 μl of the “retained phage stock” was mixed witha 150 μl aliquot of DH10B cells. The mixture was incubated at 37° C. for15 minutes. After the incubation, 700 μl of SOC medium (LifeTechnologies, Inc.) was added to the sample and the sample was thenincubated at 37° C. for an additional hour. 100 μl aliquots of thesample was spread on freshly prepared LB+Tet15 agar plates (LB agarplates containing 15 μg/ml tetracycline). The bacteria were grownovernight at 37° C.

[0082] Ten separate colonies were selected. Each colony was placed in aseparate tube containing 3 ml of LB medium. These tubes were placed at37° C. overnight. Cosmid DNA was isolated by the mini-prep method asdescribed in Maniatis et al., Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1982). Confirmationof the cosmid DNA was carried out by digesting the cosmid DNA with PstIaccording to manufacturers instructions. The average size of the genomicinserts was 20 kb or larger.

[0083] The remaining bacteria from the 10 plates were pooled andsuspended in 20 ml CG medium (CIRCLE GROW, Bio101, Inc., Cat #cp-1000C). After pooling of the bacteria, glycerol was mixed with thesuspended bacteria to a final glycerol concentration of 10% glycerol(v/v). The mixture was divided into 5 ml aliquots and stored at −70° C.as the cosmid library glycerol stock for future use.

[0084] The number of clones in a cosmid library was calculated byremoving one of glycerol stocks and then thawing the stock on ice. Thenumber of bacterial cells was determined by removing a 100 μl aliquotfrom the thawed stock and diluting that aliquot based on the expectednumber of bacterial cells (10¹¹ cells/ml). Based on the expected numberof cells, 100 μl of the sample diluted 10⁴, 10³, and 10² fold wereplated on three LB+Tet15 agar plates (LB agar plates containing 15 μg/mltetracycline) and grown at 37° C. overnight. From the plated samples, itwas possible to calculate that the cosmid library was 10¹¹ cells/ml inits glycerol stock.

EXAMPLE 7 Selection of Stability Cosmid Clones

[0085] A 20 μl aliquot of the BRL 3433 cosmid library (initial viablecell count 1.0×10⁵ cells/ml) was used to inoculate 5 ml of 15/10 medium(1.0% (w/v) Bacto Tryptone, 1.5% (w/v) Bacto Yeast Extract, 10 mM NaCl,2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 0.001% polyethelene glycol) and100 ,g/ml kanamycin. The bacteria were incubated at 30° C. for 18 hours.The bacteria were harvested by centrifuging the bacteria at 3,000 rpmfor 10 minutes at 4° C. The bacteria were then resuspended in 0.5 mlcold CCMB80 buffer (80 mM CaCl₂, 20 mM MnCl₂, 10 mM MgCl₂, 10 mMpotassium acetate, and 10% (v/v) re-distilled glycerol), stored on icefor 20 minutes and then frozen in a dry ice ethanol bath. The cells werethen thawed prior to storage at 4° C. The viable cell count of thebacterial cells was determined by diluting the cells in 0.85% NaCl andplating those dilutions on LB plates. The plates were then incubated at37° C. overnight and the colony count was determined.

[0086] After storing aliquots of the cells for 36 days at 4° C., theviable cell count of stored cosmid library clones was determined.Viability declines from an initial value of 2.5×10⁸ cells/ml to 1.6×10⁵cells/ml. Individual colonies that survived 36 days at 4° C. wereisolated by diluting the stored cosmid clones. The diluted cosmid cloneswere plated on LB plates and then incubated overnight at 37° C.Individual colonies were randomly selected. Cosmid DNA was purified fromthe isolated colonies. The cosmid DNA was analyzed by digesting withPstI, according to the manufacturer's instructions (Life Technologies,Inc.) and three isolates were retained for additional analysis andlabeled cosmid clones 1 (NRRL B-21550, deposited Mar. 28, 1996,Agricultural Research Service Culture Collection (NRRL), 1815 NorthUniversity Street, Peoria, Ill. 61604, USA), 2 (NRRL B-21551, depositedMar. 28, 1996, Agricultural Research Service Culture Collection (NRRL),1815 North University Street, Peoria, Ill. 61604, USA), and 4 (NRRLB-21552, deposited March 28, 1996, Agricultural Research Service CultureCollection (NRRL), 1815 North University Street, Peoria, Ill. 61604,USA).

EXAMPLE 8 Characterization of Cosmids Clones 1, 2 and 4

[0087] DH10B bacteria containing either cosmid clone 1, 2, 4 or cosmidvector pCP13 were incubated in 2 mls of 15/10 medium for 16 hours at atemperature of 30° C. From these cultures, a 0.3 ml aliquot was used toinoculate 50 ml of 15/10 medium and kanamycin at 10 μg/ml in 500 mlbaffled shake flasks. The flasks were incubated at 30° C. in a shakingincubator at 275 rpm until the optical densities of the cultures werebetween 0.335 and 0.399 (O.D.₅₅₀) The bacterial cultures were harvestedby centrifugation. The resultant pellets were resuspended in 4 ml ofcold CCMB80 buffer and kept on ice for 20 min. 250 ml aliquots of theresuspended samples were placed in 1.0 ml Nunc cryovials and those tubeswere then frozen in a dry ice/ethanol bath. Aliquots of these cells wereassayed for transformation efficiency according to the method of Hanahan(J. Mol. Biol. 166: 557-580 (1983) and viable cell counts were made. Thevials were then placed at −20° C. for the stability study. The remainingaliquots were thawed and retained at 4° C. for additional analysis.

[0088]FIG. 3 shows that the viable cell count of DH10B containing thevector pCP13 decreased over time at 4CC from approximately 5.0×10⁸cells/ml to approximately 1.0×10⁵ cells/ml over a period of 40 days. Incontrast, DH10B containing cosmid clones 1, 2 or 4 were more stable andonly showed a decrease of approximately 50 fold in the viable cell countover the same period of time.

[0089]FIG. 4 shows that the viable cell counts of DH10B cells containingvector pCP13 decreased 1000 fold over a period of 4 months when thecells are stored at −20° C. In contrast, DH10B cells containing cosmidclone 1 show a decreased viability of 50 fold over the same period. FIG.4 also demonstrated that DH10B cells containing cosmid clones 2 or 4showed a 10 fold improvement in survival compared to DH10B pCP13 cellsand that cosmid clone 1 improved the viable cell count 100 fold.

EXAMPLE 9 Loss of Cosmid 1 Results in a Loss of Low TemperatureStability and Retransformation of DH10B with Clone 1 Restores LowTemperature Stability

[0090] The cosmid clones were cured from the DH10B cells usingcoumermycin, as described by Danilevskaya and Gragerov, Mol. Gen. Genet.178: 233-235 (1980), herein incorporated by reference. A 20 μl aliquotof DH10B cosmid clone 1, DH10B cosmid clone 2 and DH10B cosmid clone 4cells stored at 4° C. was inoculated into 1 ml SOB—Mog medium (LifeTechnologies, Inc.). The cells were grown at 37° C. for 16 hours. Thecells were diluted to approximately 1.0×10⁴ cells/ml in SOB—Mg medium.One ml aliquots of the cells were cultured in the presence of 0, 1, 2,3, and 4 μg/ml coumermycin for 16 hours at 37° C. The cultures whichwere grown in the presence of 4 μg/ml coumermycin were then streaked onLB plates and the colonies were screened for the ability to grow in thepresence and absence of kanamycin. Cells which shoved no growth in thepresence of kanamycin have lost cosmid clone 1.

[0091] One kanamycin sensitive colony from each culture was then grownin 5 ml of 15/10 medium at 30° C. for 4 hours. The bacterial cells werecentrifuged at 3,000 rpm for 10 minutes and resuspended in 0.4 ml ofcold CCMB80 buffer. The cells were kept on ice for 20 minutes and thenfrozen in a dry ice ethanol bath. After freezing, the cells were thawedand placed at 4° C. Viable cell counts were taken at day 0, day 5 andday 11.

[0092]FIG. 4B shows that the viable cell count of cells cured withrespect to cosmid clone 1, clone 2 or clone 4 decreased as rapidly asDH10B cells or DH10B cells containing pCP13 and failed to display thestability at 4° C. seen in the cultures containing cosmid clones 1, 2,or 4. Curing cosmid clone 1 from the DH10B cells resulted in the rapidloss of viability when the cells were placed in CCMB80 buffer at 4° C.

[0093] To assess whether the reintroduction of cosmid clone 1 into thecured DH10B cells would restore stability, competent cells of DH10Bwhich had been cured of cosmid clone 1 were prepared.

[0094] Cosmid clone 1 as well as cosmid pCP13 were then retransformedinto competent cells of the DH10B strain which had been cured of cosmidclone 1 using the method of Hanahan (J. Mol. Biol. 166: 557-580 (1983)).Max efficiency DH10B competent cells lot FA4104 (Life Technologies,Inc.) were also transformed with cosmid clone 1 and pCP13. Thetransformed cultures were plated on LB plates containing 50 μg/mlkanamycin and incubated at 30° C. Competent cells of DH10B containingpCP13 and DH10B containing cosmid clone 1 as well as competent cells ofDH10B were prepared as follows: DH10B containing vector pCP13 and DH10Bcontaining cosmid clone 1 were grown in 50 ml or 15/10 medium containing50 μg/ml kanamycin until the cells were at an O.D.₅₅₀ of between 0.244and 0.258. DH10B cells were grown in 15/10 medium without kanamrycin.The cells were harvested by centrifugation and were then resuspended in4 ml cold CCMB80 buffer. After resuspension, the cells were kept on icefor 20 min. The cells were then frozen in a dry ice ethanol bath, thawedand placed at 4° C.

[0095] Viable cell counts were determined at intervals for the cellsstored in CCMB80 buffer at 4° C. FIG. 5 shows that the viable cell countof DH10B cells or DH10B containing the vector pCP13 cells decreasedrapidly from approximately 1.0×10⁸ cells/ml to approximately 1.0×10⁵ to1.0×10⁶ cells/ml over a period of approximately 16 days. On the otherhand the presence of cosmid clone 1 resulted in a 100 fold increase inthe number of viable cells/ml. These results also indicate that thereintroduction of cosmid clone 1 into DH10B cells cured of cosmid clone1 (which are unstable at 4° C.) again improves stability of these cellsat 4° C. FIGS. 6A and 6B show that the introduction of cosmid containingstability genes for storage in low temperature into DH5α (and STBL2(Trinh et al., Focus 16: 78-80 (1994) cells enhanced viability of thosecell lines at 4° C.

EXAMPLE 10 Increases in Transformation Efficiency Due to Presence ofClone 1

[0096]FIG. 7 shows that DH10B strain containing cosmid clone 1 stored at−20° C. for three months exhibits an enhanced transformation efficiency(greater than 100 fold) compared with DH10B containing the vector pCP13similarly stored. After 3 months at −20° C., DH10B containing the vectorpCP13 cells exhibited a transformation efficiency of less than 1.0×10⁵Transformants/μg. In contrast, DH10B cells containing cosmid clone 1exhibited a transformation efficiency of >1.0×10⁶ Transformants/μg.

EXAMPLE 11 Isolation of DNA Fragments of Clone 1 Responsible forIncreased Viability and Transformation Efficiency

[0097] Cosmid Clone 1 contains a 22Kb insert which was shown tosubstantially improve low temperature stability in several strains of E.coli. The 22 Kb insert in cosmid clone 1 was digested using therestriction endonuclease PstI and 2 fragments of 14Kb and 8Kb wereisolated. These two fragments were subsequently cloned into the plasmidvector pDELTA2 (the plasmid vector utilized in the generation of nesteddeletions described below) at its PstI site. The restriction maps ofboth subclones were deduced by digesting the plasmid DNA with eitherHindIII or EcoRI. These two subclones were designated pDELTA2 14Kb andpDELTA2 8Kb respectively, based on their size. To distinguish theorientation of pDELTA2 14Kb and pDELTA 8Kb, the plasmids having theopposite orientations were designated pDELTA2 14Kb+ and pDELTA1 14Kb−and pDELTA2 8Kb+ and pDelta2 8Kb−.

EXAMPLE 12 Subcloning 14 and 8Kb Fragments of Clone 1 into pDELTA2

[0098] To determine which of the subclones is able to stabilize DH10Bcells stored at 4° C. or −20° C., the pDELTA2 plasmid containing the14Kb subclone in either orientation (pDELTA2 14 Kb+ and pDELTA2 14 Kb−)and the pDELTA2 plasmid containing the 8 Kb subclone in eitherorientation (pDELTA2 8 Kb+ and pDELTA2 8Kb−) were transformed intocompetent cells of E. coli strain DH10B. Transformants were selected onLB plates containing 50 μg/ml kanamycin at 30° C. Single colonies ofDH10B cosmid clone 1, DH10B pDELTA2, DH10B pDELTA2 8 Kb+, DH10B pDELTA28Kb−, DH10B pDELTA2 14 Kb+, and DH10B pDELTA2 14Kb− were picked from theLB kanamycin plate into 2 ml of 15/10 medium containing 50μg/mlkanamycin and the cultures were incubated with shaking at 30° C. for 16hours. 0.3 ml of the overnight cultures were inoculated into 60 ml ofthe same medium and the cultures were incubated at 30° C. 275 rpm. Theoptical density was monitored and at an OD 550 nm of approximately 0.3(0.26 to 0.307), 50 ml of the cell culture was collected bycentrifugation for 10 min at 4° C. in an IEC clinical centrifuge. Thecell pellets were resuspended in 4 ml of cold CCMB80 buffer and thecells were allowed to remain on ice for 20 min. 250 μl aliquots werevialed in chilled NUNC cryovials and the vials were frozen in a dry iceethanol bath. The vials were stored at −70° C. until assay and then thevials were shifted to a −20° C. freezer for the stability study. Theremainder of the cells (approximately 2 ml) in CCMB80 buffer were frozenin a dry ice ethanol bath, thawed on ice for 10 min and assayed for theviable cell count. The remainder of the cells were then stored at 4° C.for the stability study. At intervals vials were removed from the −20°C. freezer for determination of the viable cell count. In addition, thecells stored at 4° C. were also assayed for viable cell count. Theresults are presented in FIGS. 8 and 9. The data presented in FIG. 8indicate that the 14 Kb subclone in either orientation (plasmids pDELTA214Kb+ and pDELTA2 14Kb−) substantially improves the stability of theDH10B strain when stored at 4° C. On the other hand the 8 Kb subclonedoes not improve the stability of the cells. In addition, the 14 Kbsubclone in either orientation also improves the stability of the cellsstored at −20° C. (FIG. 9). This finding demonstrates that the stabilitygene found in cosmid clone 1 resides on the 14 Kb subclone.

EXAMPLE 13 Generation of Deletion Factory Derivatives to Localize theDNA Fragments Responsible for Stability

[0099] To narrow the region in plasmid pDELTA2 14Kb+ which results ingreater stability of DH10B cells stored at 4 C., the Life TechnologiesInc. (Rockville Md.) Deletion Factory™ System was utilized. This systemgenerates a series of nested deletions of varying length in the 14Kbinsert in plasmid pDELTA1 14Kb+. The Deletion Factory System was usedfollowing the recommendations of the manufacture.

[0100] The clones were selected based on their resistance to ampicillinand sucrose or their resistance to kanamycin and streptomycin.Thirty-six different clones were selected from each selection and theplasmid DNA was isolated based on the same procedure as for cosmidclone1. The plasmid DNA from all 72 different clones for both selectionswere digested by PstI. This digestion linearized the plasmid DNA withoutcutting out the insert from the vector because one of the PstI cloningsites was lost during the generation of the deletion clones. Thelinearized plasmid DNA was separated on a 1% agarose gel with the 1kbladder (Gibco/BRL) as a standard. The size of each clone was calculatedbased on the 1kb ladder following Sambrook et al. 1989. A representativesample of the clones, based on the size of the insert DNA, were selectedfor further analysis of their ability to stabilize DH10B cells stored at4° C.

[0101] Eleven deletion derivatives of plasmid pDELTA2 14Kb+ in strainDH10B which had been generated on agar plates containing 5% sucrose and100 μg/ml ampicillin were picked into 2 ml of 15/10 medium containing100 μg/ml ampicillin and the cultures were grown for 16 hours at 30° C.DH10B containing plasmid pDELTA2, DH10B containing plasmid pDELTA 14Kb+and DH10B containing plasmid pDELTA2 14Kb− were also grown in the samemedium. 25 μl of the overnight cultures were inoculated into ml of thesame medium in a 50 ml Falcon tube and the tubes were shaken at 30° C.275 rpm for 3 hours. The cells were collected by centrifugation for min4° C. 2500 rpm in an IEC HN SII centrifuge. The cell pellets wereresuspended in 400 μl of cold CCMB80 buffer. The cells were allowed toremain on ice for 20min. The cells were frozen in a dry ice ethanol bathfor 5 minutes and thawed on ice for 15 minutes. The viable cell countwas determined by dilution in 0.85% saline. The cells were placed at 4°C. for the stability study. At intervals, the viable cell count wasdetermined and the results are presented in FIG. 10. The data indicatethat the plasmids assort into 2 distinct classes: those which stabilizethe DH10B cells and those which do not. Among the plasmids whichstabilize DH10B cells are plasmids 16, 18, 27 and 32. Among the plasmidswhich do not stabilize the DH10B cells are plasmids 14, 19, 23, 28, 29,33 and 34. As controls the plasmid pDELTA2 does not stabilize the DH10Bcells whereas plasmids pDELTA?14Kb+ and plasmids pDELTA2 14Kb− stabilizethe DH10B cells. In particular, note plasmid 32 (hereafter referred toas pDELTA2 32). This plasmid is the smallest plasmid derived frompDELTA2 14Kb+ (using the Deletion Factory System) which is capable ofstabilizing DH10B cells at 4° C.

[0102] To assess which of the plasmids still contained the gene or geneswhich improves stability of DH10B cells stored at 4° C., 16 deletionderivatives of pDELTA2 14 Kb+ in strain DH10B which had been isolated onagar plates containing 100 μg/ml streptomycin and 50 μg/ml kanamycinwere picked into 2 ml 15/10 medium containing 50 μg/ml kanamycin andwere grown overnight at 30° C. 25 μl of the overnight cultures wereinoculated into 5 ml of the same medium in 50 ml Corning tubes (Fishercat # 05-539-6) and the tubes were shaken at 30° C. 275 rpm for 3 hours.The cells were collected by centrifugation for 10 min 4° C. 2500 rpmusing an IEC HN SII centrifuge and the cell pellets were resuspended in400 μl of cold CCMB80 buffer. The cells were allowed to remain on icefor 20 min. The cells were frozen in a dry ice ethanol bath for 5 min,thawed on ice, and the viable cell count was determined. The tubes wereplaced at 4° C. for the stability study. At intervals the viable cellcounts were determined and the results are presented in FIG. 11. Thedata again indicate that the plasmids assort into 2 distinct classes:those plasmids which improve the stability of the DH10B cells and thoseplasmids which do not. Among the plasmids which stabilize the DH10Bcells are plasmids 3, 10, 12, 16, 20, 23, and 31. Among the plasmidswhich do not stabilize the DH10B cells are plasmids 1, 2, 4, 8, 9, 21,32, 35, and 36. As above, plasmid pDELTA2 does not stabilize the DH10Bcells stored at 4° C. whereas plasmid pDELTA2 14Kb+ does stabilize thecells. In particular note plasmid 16 (hereafter referred to as pDELTA216). This plasmid is the smallest plasmid from this screen which iscapable of stabilizing DH10B cells at 4° C.

[0103]FIG. 12 combines the stability studies outlined in FIGS. 10 and 11and presents the length of the insert DNA remaining after deletion of aportion of the insert using the Deletion Factory System. FIG. 12illustrates that use of the Deletion Factory System narrows the regioncontaining the stability gene to an insert size of approximately 2.5 KB.

EXAMPLE 14 Localization and Sequence of Stability/Transformation Gene onCosmid Clone 1

[0104] Deletion clones 16 and 32 delimited a region of essentialsequence approximately 2500 bases long. Primers complementary to thevector and adjacent to the deletion ends of these clones were used tosequence into the essential region. For clone 16 primer T7-25 (CGA CTCACT ATA GGG AAC TGA TCC T)(SEQ ID NO.: 1) was used and for clone 32primer SP6-25 (GAT TTA GGT GAC ACT ATA GAG ATC C) (SEQ ID NO.: 2) wasused.

[0105] DNA sequencing was performed using the method of cycle sequencingwith [α-³⁵S]dATP (Murray, V. (1989) Nucleic Acids Res. 17, 8889, hereinincorporated by reference). The sequence obtained from clone 16 was 302bases long and the sequence obtained from clone 32 was 169 bases long. ABLAST Search was performed using BLASTN with each piece of sequence. Astrong match (P(N)=2.8×10⁻⁴⁰) was found between clone 16 sequence andthe region of DNA 141 bases downstream of the E. coli gene fabB whichencodes for beta-ketoacyl-ACP synthase I (accession number M24427).Clone 32 gave a weak match (P(N)=0.47) with a region of Haemophilusinfluenzae DNA 936 bases upstream of the fabB gene (accession numberU32775 L42023). This region in E. coli was not yet in the database. Fromthe known E. coli DNA sequence in the fabB region, sequencing primerswere designed along each strand at roughly 300 base intervals. Dyeterminator sequencing was performed using an ABI 373A Stretch Sequencer.Additional primers were designed to extend the sequence from clones 16and 32 until all sequence data were able to be assembled into onecontiguous piece. The additional sequence primers included: “from 32”CCA CAT ATC CGG GTT TTT CGC TG (SEQ ID NO: 3); “fab46” GAG GTT GGC AGGTTG TAT GGA GT (SEQ ID NO: 4); “fab470” TAT GGA GCA GGC AAT CGC TGA TG(SEQ ID NO: 5); “fab1176” CGT GAA GTG TTC GGC GAT AAG AG (SEQ ID NO: 6);“fab150” AAT GCGGCC TCC GGC ACT AAC AC (SEQ ID NO: 7); “from 16” GGT TACGGT GCG TTG GCA GGA TT (SEQ ID NO: 8); “fab1104° C.” TAT CAA CGC CAT GCATCG CCA TC (SEQ ID NO: 9); “fab68C” ACT CCA TAC AAC CTG CCA ACC TC (SEQID NO: 10); “fab796” CTG GCG GCG GCG AAG AG (SEQ ID NO: 11); “fab150”AAA TGG CTG ATC GGA CTT GTT (SEQ ID NO: 12); “fab865C” TCC GGG GTG TCGTTG TATT (SEQ ID NO: 13). Sequencher 2.0 (Gene Codes Corp.) was used toassemble the data. The sequence of the significant region of clone 1(SEQ ID NO: 14) is shown in FIG. 13.

[0106] The fabB region of E. coli strain DH10B was amplified using 1.1×Elongase Supermix (LTI) and primers at positions 39(TAAATTCGAGGTTGGCAGGTT) (SEQ ID NO: 15) and 1592 (AATCGACAAAGCGGGAAGTT)(SEQ ID NO: 16) in the M24427 sequence. The cycling conditions were 30cycles of (95° C. for 30 seconds, 55° C. for 75 seconds and 72° C. for 2minutes) followed by a single incubation at 72° C. for 10 minutes. ThePCR product was purified by digesting with Exonuclease I (Adamczky, J.J., Jr. (1995) Editorial Comment 22, 36, herein incorporated byreference.) and precipitating with isopropanol (Brow, M. A. D. (1990) inPCR Protocols: A Guide to Methods and Applications (Innis, M. A.,Gelfand, D. H., Sninsky, J. J., and White, T. J., eds.) p. 194, AcademicPress, San Diego. The pellet was redissolved in the initial volume of 10mM Tris-HCl (pH 7.5), 5 mM NaCl, 0.1 mM EDTA. Again dye terminatorsequencing was performed using an ABI 373A Stretch Sequencer and primersfrom the list above. The sequence alignment program Align Plus(Scientific and Educational Software) was used to compare the sequencesderived from clone 16 and DH10B to show that they were identical overthe region sequenced.

[0107] In the sequence from FIG. 13, the essential region of clone 1contained several open reading frames in addition to fabB. Deletionswere made to confirm that the function was correlated specifically withthe fabB gene (see below). Open reading frames greater than 100 aminoacids in the essential region of clone 1 are as follows: Number of ORFBases Amino Acids 1 (fabB) 1043-2263 406 2 1900-1133 255 3 488-24  154

[0108]FIG. 14 depicts the open reading frames and the location of theMluI restriction sites on the approximately 2500 bp essential region ofcosmid clone 1.

[0109] From the sequencing data presented above, the fabB gene can beidentified as one potential gene which stabilizes the E. coli cellsstored in CCMB80 buffer at 4° C. or −20° C.

[0110] To further evaluate the ability of the fabB gene to enhance thestability of E. coli strains stored at −20° C., the fabB gene frompDELTA2 32 was subcloned into plasmid pDELTA2. Previous data (Tsay J. etal. J. Bact 174: 508-513 1992) indicated that the entire fabB gene couldbe subcloned as a 1.8Kb MluI fragment. pDELTA2 32 DNA was digested withMluI at 37° C. for 2 hours. The digestion reaction was electrophoresedon a 1% agarose gel in TAE buffer and the 1.8Kb fragment was purifiedusing GlassMax (Gibco BRL) following the protocol from the manufacturer.Plasmid pDELTA2 was digested with MluI at 37° C. for 3 hours and treatedwith alkaline phosphatase at 37° C. for 1 hour. Sodium dodecylsulfatewas added to 0.5% followed by proteinase K to 50 μg/ml . The reactionwas incubated at 37° C. for 1 hour and then extracted with phenolchloroform. The DNA was precipitated and resuspended in 10 mM Tris (pH7.5) 1 mM EDTA buffer. The 1.8Kb MluI fragment was then ligated with theMluI cut alkaline phosphatase treated pDELTA2 vector DNA using T4 DNALigase at room temperature for 16 hours. The ligation reaction was thenused to transform Max Efficiency DH10B competent cells (Gibco BRL)according to the directions of the manufacturer. Colonies selected on LBplates containing 100 μg/ml ampicillin at 37° C. were then grown for 16hours 37° C. in LB medium containing 100 μg/ml ampicillin and plasmidDNA was isolated. The plasmid DNA was digested with MluI and screenedfor the presence of the 1.8Kb MluI fragment. 4 plasmids (numbered 10,13, 14 and 15) containing the 1.8Kb MluI fragment were selected forfurther study. The plasmid DNA was further screened for the orientationof the MluI fragment by digesting the plasmid DNA with BglI. Gelelectrophoresis of the digested fragments indicated that clones 10 and15 contained the MluI fragment in one orientation and clones 13, 14contained the MluI fragment in the opposite orientation.

[0111] To evaluate the ability of these plasmids containing a clonedMluI insert derived from plasmid pDELTA2 32 to encode the enzymaticfunction characteristic of fabB gene product, the plasmids designatedclones 10, 13, 14 and 15 (as well as plasmids pDELTA2 32 and pDELTA2)were introduced into an E. coli strain which contains a mutation in thefabB gene. Specifically the strain contains the fabB15(ts) allele whichencodes a temperature sensitive fabB gene product. The mutation resultsin the ability of the strain to grow at 30° C. but not at 42° C. Thestrain was obtained from the E. coli Genetic Stock Center at YaleUniversity and was given the designation CGSC5641. Competent cells ofthis strain were prepared as follows: Several colonies of CGSC5641 werepicked from an LB plate grown at 30° C. into 50 ml of SOB medium and theflask was shaken at 30° C. 275 rpm.

[0112] When the optical density at 550 nm reached 0.308 the cells werecollected by centrifugation for 10 min at 4° C. 2500 rpm in an EEC HNSII centrifuge. The cell pellet was resuspended in 4 ml cold CCMB80buffer and the cells were placed on ice for 20 min. 250 ul of the cellswere aliquoted into chilled NUNC tubes and the tubes were frozen in adry ice ethanol bath. The vials were stored at −80 C. Several tubes ofthe competent cells were removed from the −80 C. freezer and were thawedon ice for approximately 15 min. 100 ul of the competent cells weretransformed with 5 ul of plasmid DNA: pDELTA2, pDELTA2 32, clone 10,clone 13, clone 14, clone 15 according to published procedures (HanahanJ. Mol. Biol. 166: 557-580 (1983)). The cells were expressed at 30° C.for 1 hour after addition of SOC. After the expression period, 100 μl ofthe cells were plated on LB plates containing 100 μg/ml ampicillin andthe plates were incubated at either 30° C. or 42° C. If colonies appearon the plates at both 30° C. and 42° C. then the plasmid has providedthe enzymatically active fabB gene product which is defective at 42° C.in the E. coli strain CGSC5641. If colonies only appear on theampicillin plates at 30° C., then the plasmid has failed to provide theactive fabB gene product. Table 2 shows the results of thecomplementation of the fabBts mutation. TABLE 2 Number of AmpicillinResistant Colonies Strain 30° C. 42° C. pDELTA2 130 109  0  0 Clone 32 24  22  23  31 Clone 10 162 207 151 160 Clone 13 185 181 179 150 Clone14 147 152 137 132 Clone 15 205 244 207 213

[0113] The results in Table 2 indicate that transformation with thepDELTA2 plasmid only results in ampicillin resistant colonies at 30° C.indicating that the pDELTA2 plasmid does not encode an active fabB geneproduct. Transformation with plasmid pDELTA 32, clone 10, clone 13,clone 14 and clone 15 results in ampicillin resistant colonies at both30° C. and 42° C. indicating that these plasmids encode a wild typ fabBgene product. Therefore clones 10, 13, 14, and 15which contain insertDNA of 1.7Kb (including 1.2 KB of the coding sequence of fabB) expressan active fabB gene product. These clones are hereafter referred to aspDELTA2 fabB10, pDELTA2fabB13, pDELTA2fabB14 and pDELTA2 fab15.

[0114] To evaluate the ability of these plasmids designated pDELTA2fabB10, pDELTA2 fabB13, pDELTA2 fabB14 and pDELTA2fabB15 to improve thestability of DH10B cells stored at 4° C. and −20° C., 5 μl of each ofthe plasmids were transformed into Max Efficiency DH10B competent cells(lot# HFK701) according to directions provided by the Manufacturer.Plasmids pDELTA2 and pDELTA2 32 were also transformed as controls.Transformants were selected on LB agar plates containing 100 μg/mlampicillin at 30° C. The next day one ampicillin resistant transformantfrom each transformation was picked into 2 ml of 15/10 medium containing100 μg/ml ampicillin and the cultures were shaken for 16 hours at 30° C.0.25ml of each overnight culture was inoculated into 60 ml of the samemedium in a 500ml baffled shake flask and the cultures were shaken at30° C. 275 rpm in a New Brunswick floor shaker. The optical density wasmonitored at 550 nm and the cultures were harvested when the opticaldensity reached 0.26-0.32. 50 ml of the cultures were centrifuged for 10min at 4° C. in an IEC bench top centrifuge and the pellets wereresuspended in 4 ml of cold CCMB80 buffer. The cells were incubated onice for 20 min. The cells were divided into 2 2 ml portions. One 2 mlportion was frozen in a dry ice ethanol bath for 5 minutes. The cellswere then thawed on ice. The viable cell count was determined usingserial dilution in 0.85% NaCl. The cells were then placed in a 4° C.refrigerator. The second portion of the cells was processed as follows:250 ul of the cells were vialed in NUNC cryovials and the vials werefrozen in a dry ice ethanol bath. One tube of cells was used todetermine the viable cell count and the remainder of the vials wereplaced in a −20° C. freezer. At intervals the viable cell count wasdetermined from cells stored at 4° C. In addition, tubes were removedfrom storage at −20° C. for determination of the viable cell count. Theresults are presented in FIGS. 15 and 16. As seen in FIG. 15 the viablecell count for DH10B cells containing the plasmid PDELTA2 decreased 100fold after 9 days storage at 4° C. The viable cell count of the DH10Bcells containing plasmid pDELTA2 32 (the source of the MluI fragmentused in the cloning of the fabB coding sequence) decreased only 8 foldover the same period of time and thus resulted in a 30 fold enhancementin the viable cell count compared too DH10B cells containing the plasmidpDELTA2. DH10B cells containing the pDELTA2 fabB clones 10, 13, 14, and15 also showed enhance survival relative to DH10B cells containingvector pDELTA2 by 30-60 fold. Therefore the clones containing the fabBcoding sequence resulted in enhanced survival of DH10B cells at 4° C.

[0115] As seen in FIG. 16, both plasmids pDELTA2 32 and pDELTA2fabBclones 10, 13, 14, and 15 also result in enhanced survival of DH10Bcells when the cells are stored at −20° C. The fabB clones resulted in a400-700 fold enhancement in the viability of DH10B cells relative to theviability of the DH10B cells containing the plasmid pDELTA2.

[0116] In order to provide more evidence that plasmids which express anactive fabB gene product enhance stability of DH10B cells stored at 4°C. and −20° C., the coding sequence of fabB on plasmids pDELTA2 fabB 14and pDELTA2 fabB 15 was interrupted by deletion of an approximately 750bp fragment. Plasmid DNA from pDELTA2 fabB 14 and pDELTA2 fabB13(encoding an enzymatically active fabB gene) were digested with Kpn2I at55° C. for 1 hour. PinAI was then added and the reaction was incubatedfor a further 1 hour at 37° C. The digestion reaction waselectrophoresed on a 1% agarose gel with TAE buffer. A 9Kb fragmentisolated from the gel was purified with GlassMax (Gibco BRL) and thepurified DNA was resuspended in TE buffer. The ends of the 9Kb fragmentwere ligated with T-DNA ligase at room temperature for 16 hours. Theligation reaction was used to transform Max Efficiency DH10B competentcells with selection on LB plates containing 100 ,g/ml ampicillin at 37°C. Colonies were picked from the plate into LB medium containing 100μg/ml ampicillin and the cultures were grown for 16 hours at 37° C.Plasmid DNA was isolated, digested with MluI and BglI and analyzed bygel electrophoesis. Two plasmids, designated pDELTA2 14 deletion andpDELTA2 15 deletion, were shown to be lacking the 750 bp fragment.

[0117] To determine if deletion of the 700 bp fragment resulted in lossof the fabB gene product, plasmids pDELTA2, pDELTA2 fabB14, pDELTA2fabB15 pDELTA2 14 deletion and pDELTA2 15 deletion were transformed intocompetent cells of CGSC5641 (fabB15ts). The results are presented inTable 3. TABLE 3 Number of Ampicillin Number Resistant Colonies NumberTemperature Clone 42° C. 30° C. Screened Resistant fabBts  0  0 — —+pDELTA2  1 123 20  0 +clone 14 114 123 20 20 +clone 15 288 360 20 20 14deletion  0  56 20  0 15 deletion  0 221 20  0

[0118] Table 3 shows the results of the transformation of CGSC5641(fab15s) competent cells with pDELTA2, pDELTA2 fabB14, pDELTA2 14deletion, pDELTA2 fabB15 or pDELTA2 15 deletion plasmid. Transformationof plasmid pDELTA2 into competent cells of CGSC5641 results inampicillin resistant colonies only at 30° C. indicating that the pDELTA2plasmid does not encode a functional fabB gene product.

[0119] Transformation of plasmids pDELTA2fabB 14 or pDELTA2fabB15 intocompetent cells of CGSC5641 result in ampicillin resistant colonies atboth 30° C. and 42° C. indicating that plasmids pDELTA1 fabB14 andpDELTA2fabB15 encode a functional fabB gene product. Transformation ofplasmids pDELTA2 14 deletion or pDELTA2 15 deletion result in ampicillinresistant colonies only at 30° C. indicating that these plasmids do notencode a functional fabB gene product. Therefore, deletion ofapproximately 750 bp of the fabB coding region results in an inabilityto complement a fabBts mutation.

[0120] MluI sites are at positions 768 and 2436. The deletions inMluI14Δ and MluI15Δ were confirmed by sequencing across the juncturewith the fab470 primer (SEQ ID NO: 5). The 3′ end of the fab470 primeris positioned at 1298 bases in the our fabB gene sequence. The deletionsshould remove bases 1310 through 2033. Typically, approximately thefirst twenty bases of sequence 3′ of the primer are not readable, sousing this primer the first readable base would be at about 1318 in thefabB sequence if there were no deletion, and at about 2041 if thedeletion was present. Since the readable sequence begins at 2041 forboth clones the deletions are as expected. The Sequence of clonesMluI14Δ and MluI15Δ from primer fab470 (SEQ ID NO.: 17) are as follows:  ²⁰⁴¹CTCTCTGGGCGCTGCTGGCGTACAGGAAGCTATCTACTCTCTGCTGATGCTGGAACACGGCTTTATCGCCCCGAGCATCAACATTGAAGAGCTGGACGAGCAGGCTGCGGGTCTGAACATCGTGACCGA²¹⁶⁹

[0121] To determine whether disruption of the fabB coding sequencereduces the ability of the plasmids to stabilize DH10B cells, theplasmids pDELTA2, pDELTA2 fabB14, pDELTA2 fabB15, pDELTA2 14 deletion,and pDELTA2 15 deletion were transformed into Max Efficiency DH10B lotHFK701. The transformants were plated on LB plates containing ampicillinat 100 μg/ml at 30° C. for 16 hours. Single colonies from eachtransformation were picked into 2 ml 15/10 medium containing ampicillinat 100 μg/ml and the cultures were shaken at 30° C. for 16 hours. 0.25ml of the overnight culture were inoculated into 50 ml of the samemedium and were grown to an optical density at 550 nm of 0.27 to 0.30.The cells were collected by centrifugation for 10 min 4° C. 2500 rpm inan EEC HN SII centrifuge and the cell pellet was resuspended in 4ml coldCCMVB80 buffer. The cells were placed on ice for 20 min and 250μl ofcells were vialed in chilled NUNC cryovials. The cells were frozen for 5min in a dry ice ethanol bath and were placed in a −20° C. freezer. Theremainder of the cells were frozen in a dry ice ethanol bath, thawed andplaced at 4° C. Viable counts were determined by serial dilution in0.85% saline. At intervals viable counts were determined after variousperiods at either 4° C. or −20° C.

[0122] The results are presented in FIGS. 17 and 18. FIG. 17 shows thatthe viable cell count of DH10B cells containing the pDELTA2 plasmidstored at 4° C. in CCMB80 buffer declines over a period of 30 days from1×10 ⁸ cells/ml to 1×10⁵ cells/ml. The viable cell count of cellscontaining plasmid pDELTA2 fabB 14 or pDELTA2 fabB15 is approximately100 fold higher than cells containing the pDELTA2 vector. These resultsindicate that a plasmid which contains a functional fabB genesignificantly enhances the stability of DH10B cells stored at 4° C.DH10B cells containing deletion derivatives which interrupt the fabBcoding sequence (plasmids pDELTA2 14 deletion and pDELTA2 15 deletion)have a substantially lower viable cell count than in DH10B cellscontaining an intact fabB coding sequence. These results indicate thatdisruption of the fabB coding sequence eliminates the ability of theseplasmids to stabilize DH10B cells. The viable cell counts of DH10BpDELTA2 14 deletion and pDELTA15 deletion are slightly higher than theviable cell counts of DH10B pDELTA2 cells. These results may indicate aslight increase in the fabB gene product (from the intact fabB gene inthe chromosome of strain DH10B) in cells which contain plasmids withinterrupted fabB coding sequence possibly due to regulatory effects ofthese plasmids. FIG. 18 indicates that DH10B cells containing pDELTA2plasmid stored at −20° C. for 60 days show a marked instability with a 3log decrease in viable cell count. The presence of a functional fabBgene product (due to the presence of plasmids pDELTA2fabB14 orpDELTA2fabB15) results in a 100 fold increase in the viable cell count.Again, the presence of deletion derivatives which interrupt the fabBcoding sequence results in a slightly higher viable cell count than incells containing plasmid pDELTA2 but a much lower viable cell count thancells containing plasmids which encode a functional fabB gene product.

[0123] These results indicate that the presence of an intact fabB geneon a multicopy plasmid improves the stability of DH10B cells when thecells are stored at either 4° C. or −20° C.

EXAMPLE 15 Effect of FabB on Levels of Unsaturated Fatty Acids

[0124] One consequence of the cloning and overexpression of the fabBgene in E. coli is an increase in total amount of unsaturated fattyacids found in membrane phospholipids. This increase is a result of anincrease in the cis vaccenate level (de Mendoza D. et al. J. Biol Chem258: 2098-2101 (1983)). To evaluate whether the cloned fabB gene causesa similar increase in the cis-vaccenate level (and thus an increase inthe total amount of unsaturated fatty acids) in DH5α and DH10B cells,pCP13, cosmid clone 1, pDELTA2 and pDELTA2fabB15 were transformed intocompetent cells of DH5α and DH10B and the cells were grown for ananalysis of the fatty acids present in the cell membrane. Cells werealso analyzed for stability at −20° C. in CCMB80 buffer. Transformantswere selected at 30° C. on LB plates containing 50 μg/ml kanamycin (forpCP13 and cosmid clone 1) or 100 μg/ml ampicillin (for pDELTA2 andpDELTA2 fabB15). Colonies from these plates were inoculated into 2 ml ofmedium. For DH5α pCF13 and DH5α cosmid clone 1 the cells were inoculatedinto SOB medium containing 50 μg/ml kanamycin. For DH5α pDELTA2 and DH5αpDELTA2 fabB15 the cells were inoculated into SOB medium containing 100μg/ml ampicillin. For DH10B pCP13 and DH10B cosmid clone 1 the cellswere inoculated into 15/10 medium containing 50 μg/ml kanamycin. ForDH10B pDELTA2 and DH10B pDELTA2 fabB15 the cells were inoculated into15/10 medium containing 100 μg/ml ampicillin. The cells were grown at30° C. for 16 hours. 250 μl of the cultures were inoculated into 60 mlof the same medium and the cultures were grown at 30° C. 275 rpm. Theoptical density was monitored at 550 nm and when the cultures reached anoptical density of approximately 0.3 (range 0.253-0.312) the cells werecollected by centrifugation. Specifically, 40 ml of the cells werecollected by centrifugation for 10 min at 4° C. 2500 rpm in an IEC HNSII centrifuge. The cell pellets were resuspended in 3.2 ml of coldCCMB80 and the cells were placed on ice for 20 min. 250 μl of the cellswere vialed in NUNC cryovials and the vials were frozen in a dry iceethanol bath for 5 min. The cells were stored in a −20° C. freezer forthe stability study. At intervals 2 vials were removed from the freezerand the cells were assayed for the viable cell count. The remainder ofthe cells (15-20 ml) were collected by centrifugation, washed once in 10mM Tris HCl pH7.5 and the cell pellets were stored at −20° C. The lipidswere extracted from the cell pellets and were analyzed for fatty acids.

[0125] The cell pellet was washed and resuspended in distilled water andthe solution was transferred to a glass tube. Two volumes of methanoland one volume of chloroform were added to the glass tube for eachvolume of cell solution. After mixing and centrifuging, the supernatantwas transferred to a new glass tube. One volume of distilled water andone volume of chloroform were added to the tube and a two phase solutionwas obtained. The organic phase was transferred to a new glass tube andthe organic solution was dried by blowing nitrogen gas onto the surfaceof the solution. 2 ml of ethanol was then added to each tube and thesolution was again dried with nitrogen. The ethanol wash step wasrepeated. 2 ml of 0.5M sodium methoxide (Aldrich) was added to each tubeand the tubes were kept at room temperature for 1 hour. 0.1 ml of lacialacetic acid and 5 ml of distilled water were added to each tube. Thesolution was extracted 2 times with 5 ml hexane. The solution was thendried and redissolved in 200 μl of carbon disulfide.

[0126] For GC analysis, a Hewlett-Packard 5890 Series II gaschromatograph equipped with a capillary inlet system and HP 7673automatic sampler was used (Hewlett-Packard, Palo Alto, Calif.). Thecolumn was a 30 m×0.32 u m I.D. fused silica capillary column coatedwith demethylpolysiloxane with a film thickness of 1.0 u (Alltech,Deerfield, Ill.). Ultra high purity helium at a flow rate of 1 ml/minwas used as the carrier gas. A split-injection (50:1) mode was used,with the injector set at 285° C. The oven temperature was set at 190° C.After each analysis, the oven temperature was increased by 50° C./min to285° C. and held for 10 min. The FID detector temperature was 285° C.

[0127] Table 4 shows the effects of the presence of fabB clones on thelevel of unsaturated fatty acids in DH5α and DH10B. TABLE 4 % Survival−20° C. 1 2 Fatty Acid Composition # Samples Mo Mo C14:0 C16:0 C16:1C17:0 C18:0 C18:1 C19:0 1 DH5α pCP13 6 2 2 41.5 32.5 3.8 3.2 15.5 1.5 2DH5α clone 1 28 9 1.8 22.2 25.5 4.5 3.6 41 1.4 3 DH5α 7 1 0.3 43.2 37.32.4 1.4 12.2 3.2 pDELTA2 4 DH5α 28 12 0.2 4.8 22 0.3 3.4 69.2 0.1 pDELTAfabB15 5 DH10B pCP13 7 0.7 0.15 34.6 30.4 4.2 0.4 29.9 0.3 6 DH10B clone1 6 4 1.6 28.5 26.2 4.1 0.4 40.6 0.05 7 DH10B 4 0.4 1.6 35.9 27.4 5.40.3 29.3 0.1 pDELTA2 8 DH10B 59 29 0.3 14.2 17.6 3.5 1.9 62.4 0.1 pDELTAfabB15

[0128] In Table 4, C14:0 refers to myrsitic acid, C16:0 refers topalmitic acid, C16:1 refers to palmitoleic acid, C17:0 refers tomargaric acid, C18:0 refers to stearic acid, C18:1 refers tocis-vaccenic acid, and C19:0 refers to nondecylic acid. As seen in Table4, and FIG. 19, the presence of cosmid clone 1 in strain DH5α increasesthe cis vaccenate level from 15.5% in strain DH5α pCP13 to 41% in DH5αcosmid clone 1. The presence of plasmid pDELTA2 fabB15 increases the cisvaccenate level from 12.2% in DH5α pDELTA2 to 69.2% in DH5αpDELTA2fabB15. A similar increase in the cis vaccenate level is seen instrain DH10B. The cis vaccenate level increases from 29.9% in strainDH10B pCP13 to 40.6% in strain DH10B cosmid clone 1 and from 29.3% instrain DH10B pDELTA2 to 62.4% in strain DH10B pDELTA2 fabB15. When thelevel of total unsaturated lipids (both C18:1 and C16:1) are calculated,the presence of a functional fabB gene on a plasmid increases theunsaturated fatty acid level from 48-49% in strains DH50α pCP13 and DH5αpDELTA2 to 66.5% in DH5α cosmid clone 1 and to 91% in strain DH5αpDELTA2 fabB15 (FIG. 20). As expected, the higher copy number plasmidpDELTA2 fabB15 increases the unsaturated lipid level more than the lowercopy number cosmid clone 1. In strain DH10B the presence of a functionalfabB gene on a plasmid increases the unsaturated fatty acid level from56-60% in strains DH10B pCP13 and DH10B pDELTA2 to 67% in strain DH10Bcosmid clone 1 and to 80%o in DH10B pDELTA2 fabB15 (FIG. 20). Again, thehigher copy number plasmid pDELTA2 fabB15 increases the unsaturatedlipid level more than the lower copy number cosmid clone 1.

[0129] The results of the stability study can be found in FIGS. 21 and22. As seen in FIG. 21, the DH5u cells containing pCP13 or pDELTA2 areunstable at −20° C. and the viable cell count decreases from 3-5×10⁸cells/ml to 3.9-10×10⁶ cells/ml over a period of 2 months. DH5α cellscontaining cosmid clone 1 or pDELTA2 fabB 15 are more stable than thecontrol cells and have 5-10 fold more viable cells/ml compared to cellscontaining pCP13 or pDELTA2. Similarly DH10B cells containing pCP13 orpDELTA2 are also unstable at −20° C. decreasing from 2-3×10⁸ cells/ml toapproximately 1.0×10⁶ cells/ml over the same period of time at −20° C.(FIG. 22). DH10B cells containing cosmid clone 1 or pDELTA2,fabB 15 aremore stable with 10-40 fold higher viable cell counts than found incontrol cells.

[0130] As indicated in FIG. 23, a correlation exists between thesurvival of the DH5α and DH10B cells at −20° C. and the amount ofunsaturated fatty acids found in the cell membrane. When the results areplotted as cell survival at −20° C. on the y axis and percent of totallipids as unsaturated fatty acids on the x axis, the results indicatethat the higher the value of total unsaturated lipids in the cellmembrane, the greater the survival rate of the cells at −20° C. (FIG.23).

EXAMPLE 16 Increased Survival of SB3499B Cells

[0131] To determine if the increased survival of SB3499B cells stored at−20° C. was due to an increased level of unsaturated fatty, acids in thecell membrane, DH10B and SB3499B cells were grown at several differenttemperatures and the lipids were analyzed as in Example 15. In addition,strain CY322 containing a mutation in fabF was also included in thisstudy. This particular fabF mutation results in the overproduction ofcis vaccenate at all growth temperatures (Ulrich, A. K. et al., J. Bact154: 221-230 (1983)). Specifically, CY322, DH10B and SB3499B masterseeds stored at −70 C. were used as a source of the cells used for thisexperiment. The strains were streaked on LB plates and the plates wereincubated at 23° C., 30° C., 37° C., and 42° C.. The cells from theseplates were used to inoculate 1.5ml broth cultures. For DH10B andSB3499B the cells were inoculated into 15/10 medium. For CY322 the cellswere inoculated into SOB medium. The cultures were grown at 23° C., 30°C., 37° C., and 42° C. for 16 hours. 0.3 ml of the DH10B and SB3499Bcells were inoculated into 60 ml of 15/10 medium and the cultures weregrown at the appropriate growth temperature 273 rpm and the opticaldensity was monitored at 550 nm. 0.2 ml of the CY322 cells wereinoculated into 25 ml of SOB medium and the cultures were shaken at theappropriate growth temperature. When the optical density of the CY322cells reached 0.25 (range 0.24-0.262) the cells were collected bycentrifugation at 4° C. for 10 min 2500rpm in an IEC HN SII centrifuge.The cell pellets were washed once with 10 mM Tris HCl pH 7.5 and thecell pellets were stored at −20° C. The lipids were extracted andanalyzed by GC as presented in Example 17. For the DH10B and SB3499Bcells, the optical density was monitored and when the OD reachedapproximately 0.3 (range 0.24-0.33, 40 ml of the cells were collected bycentrifugation and the cell pellets were resuspended in 0.32 ml of coldCCMB80. The cells were allowed to remain on ice for 20 min and 250 μlwas vialed in NUNC cryovials. The vials were frozen in a dry ice ethanolbath and were stored at −20° C. for the stability study. 15-20 ml of thecells were also centrifuged and the cell pellets were washed once in 10mM Tris HCl pH7.5. The cell pellets were stored at −20° C. and were thenextracted for lipid analysis as in Example 17.

[0132] The results of the lipid analysis are found in Table 5 and FIG.24. TABLE 5 Fatty Acid Composition Growth # Sample Temp. C14:0 C16:0C16:1 C17:0 C18:0 C18:1 1 DH10B 23° C. 5.6% 41.0% 31.8% 2.1% 2.1% 17.4%3 DH10B 30° C. 4.3% 39.4% 32% 2.6% 1.3% 20.3% 5 DH10B 37° C. 3.3% 40.3%29.8% 3.7% 3.3% 19.6% 9 DH10B 42° C. 4.5% 49.2% 27.5% 2.5% 2.3% 14.0% 2SB3499B 23° C. 0.8% 24.2% 34.4% 2.3% 0.8% 37.5% 4 SB3499B 30° C. 1.3%24.4% 27.9% 3.0% 1.7% 42.1% 6 SB3499B 37° C. 1.4% 29.2% 27.5% 3.7% 0.6%37.6% 10 SB3499B 42° C. 2.9% 42.8% 28.8% 3.2% 2.9% 19.4% 11 CY322 23° C.2.4% 29.6% 28.3% 1.6% 4.9% 33.2% 12 CY322 30° C. 2.6% 32.4% 22.5% 3.9%6.0% 32.6% 13 CY322 37° C. 3.2% 33% 21.4% 6.1% 4.1% 32.3% 14 CY322 42°C. 1.6% 37.6% 14.6% 1.8% 5.7% 38.7%

[0133] In Table 5, C14:0 refers to myrsitic acid, C16:0 refers topalmitic acid, C16:1 refers to palmitoleic acid, C17:0 refers tomargaric acid, C18:0 refers to stearic acid, C18:1 refers to vaccenicacid, and C19:0 refers to nondecylic acid.

[0134]FIG. 24 indicates that cells of SB3499B have a higher level of cisvaccenate (37-42%) compared to cells of DH10B (17-20%o). The higherlevels of cis vaccenate are found in SB3499B cells grown at either 23°C., 30° C., or 37° C. However, if the SB3499B cells are grown at 42° C.the level of cis vaccenate is not appreciably greater than in DH10Bcells grown at 42° C. In addition, the cells of CY322 grown at 23° C.,30° C., 37° C., or 42° C. have levels of cis vaccenate which are greaterthan those levels found in DH10B and which do not appreciably vary withthe growth temperature. This finding is consistent with the knownphenotype of E. coli strains which have the fabF mutation (such asCY322), specifically the overproduction of cis vaccenate regardless ofgrowth temperature.

[0135]FIG. 25 also indicates that, at 23° C., 30° C., and 37° C., thetotal level of unsaturated lipids (C16:1 and C18:1) is greater inSB3499B cells than in DH10B cells. The levels range from 65% to 71% inSB3499B and from 49% to 52% in DH10B. On the other hand, when these 2strains are grown at 42° C., the levels of unsaturated lipids isessentially the same in both strains (41% in DH10B and 48% for SB3499B.These results indicate that SB3499B, at most growth temperaturesexamined, overproduces cis vaccenate compared to cells of DH10B. As aresult, at these same growth temperatures, the levels of unsaturatedlipids in the phospholipid are higher in SB3499B than in DH10B (FIG.25).

[0136] The results of the −20° C. stability study are presented in FIGS.26 and 27. The DH10B cells are quite unstable when stored at −20° C. butappear more unstable when cells are grown at 37° C. or 42° C. DH10Bcells grown at 23° C. or 30° C. and stored at −20° C. lose approximately900° C. of the viable cells after one month at −20° C. whereas DH10Bcells grown at 37° C. or 42° C. lose approximately 99% of the viablecells. SB3499B cells again demonstrate enhanced survival compared toDH10B cells but the effect depends on the growth temperature. TheSB3499B cells grown at 23° C. and stored at −20° C. for one month have a6 fold higher viable cell count compared to DH10B cells. However, theimprovement in the viable cell count of SB3499B cells relative to DH10Bcells becomes progressively less pronounced as the growth temperatureincreases. SB3499B cells grown at 37° C. or 42° C. are no more stable at−20° C. than are the DH10B cells. The correlation between totalunsaturated fatty acids in the cell membrane and cell survival at −20°C. can be observed when the cell survival at −20° C. is plotted againstthe total unsaturated fatty acid content of the cell membrane. This datais shown in FIG. 28. The data indicates a correlation in that for bothstrains SB3499B and DH10B, an increase in the total unsaturated fattyacid composition of the cell membrane results in an enhanced cellsurvival at −20° C.

[0137] While the invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications and this application is intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the inventions pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

1 17 25 base pairs nucleic acid single linear 1 CGACTCACTA TAGGGAACTGATCCT 25 25 base pairs nucleic acid single linear 2 GATTTAGGTGACACTATAGA GATCC 25 23 base pairs nucleic acid single linear 3CCACATATCC GGGTTTTTCG CTG 23 23 base pairs nucleic acid single linear 4GAGGTTGGCA GGTTGTATGG AGT 23 23 base pairs nucleic acid single linear 5TATGGAGCAG GCAATCGCTG ATG 23 23 base pairs nucleic acid single linear 6CGTGAAGTGT TCGGCGATAA GAG 23 23 base pairs nucleic acid single linear 7AATGCGGCCT CCGGCACTAA CAC 23 23 base pairs nucleic acid single linear 8GGTTACGGTG CGTTGGCAGG ATT 23 23 base pairs nucleic acid single linear 9TATCAACGCC ATGCATCGCC ATC 23 23 base pairs nucleic acid single linear 10ACTCCATACA ACCTGCCAAC CTC 23 17 base pairs nucleic acid single linear 11CTGGCGGCGG CGAAGAG 17 21 base pairs nucleic acid single linear 12AAATGGCTGA TCGGACTTGT T 21 19 base pairs nucleic acid single linear 13TCCGGGGTGT CGTTGTATT 19 2658 base pairs nucleic acid single linear 14CGTAGNTTTC GTTNCATTGG CCCTCAAACC CCTAATAGCG CCAGCGACAA CAACGCGCTG 60GCAATACCAC CGCCGATAAT CGCCGCTTCC CGTTTGCTGC TGCCCGTGCG GTTAAACCAC 120GGCGCGGAGC AGGGGAGCGG TAATGTCTGT TCCATCACCC CGCAAAGCAT TTCCCGTTTG 180CGCCCAAAGC CCTTACGTTT TTGCATCGTG AATCCGGCGT CCTGCAAACC GCGGCGGACA 240AAACCGGCAG ACGTAAATGT CGCCAGCGTG CCGCCCGGAC GCGCCAACCT TGCCATGGCG 300TTAAACAGAT TTTGCGTCCA CATATCCGGG TTTTTCGCTG GCGCAAAGCC GTCCAGAAAC 360CAGGCATCTA CTTTTTGATT TAGCGAATCG TCCAGTTGGC TGGTCAGTTC GTTAATATCG 420CCAAACCATA AATCCAGCGT CACGCGGCCT TCATCGAGCA ATAAACGATG GCAACCGGGC 480AAGGGCATTG GCCACTGCGC CTGAAGTTGT TCTGCCCACG GAGCCAGTTC CGGCCAGTGT 540TGATGCGCTA AGGCTAAATC CGCACGGGTG AGGGGAAATT TCTCAAAACT AATGAAATGT 600AAGCGTTGTA ATTGCGCTTG CGGATGCGCT TCGCGAAACT GATCAAATGC CTGCCATAGC 660GTCAGGAAGT TTAATCCGGT GCCGAAGCCG CTCTCTGCTA CCACAAACAG AGGATGTGGA 720TGCTCAGGAA AGCGTACCTC TAATTGGTTG CCTCCCAGAA AAACATAACG CGTCTCTTCC 780AGCCCGTTAT CGTTGGAAAA ATAGACATCG TCAAAATCTC GGGAAACAGG TGTACCCTCA 840GCATTAAATT CGAGGTTGGC AGGTTGTATG GAGTAGTGTT TCACGTAAGT TACTCGTCTT 900ACAGGCGGTG GCTCGATCTT AGCGATGTGT GTAAGGCTGC GCAAATTTCT CTATTAAATG 960GCTGATCGGA CTTGTTCGGC GTACAAGTGT ACGCTATTGT GCATTCGAAA CTTACTCTAT 1020GTGCGACTTA CAGAGGTATT GAATGAAACG TGCAGTGATT ACTGGCCTGG GCATTGTTTC 1080CAGCATCGGT AATAACCAGC AGGAAGTCCT GGCATCTCTG CGTGAAGGAC GTTCAGGGAT 1140CACTTTCTCT CAGGAGCTGA AGGATTCCGG CATGCGTAGC CACGTCTGGG GCAACGTAAA 1200ACTGGATACC ACTGGCCTCA TTGACCGCAA AGTTGTGCGC TTTATGAGCG ACGCATCCAT 1260TTATGCATTC CTTTCTATGG AGCAGGCAAT CGCTGATGCG GGCCTCTCTC CGGAAGCTTA 1320CCAGAATAAC CCGCGCGTTG GCCTGATTGC AGGTTCCGGC GGCGGCTCCC CGCGTTTCCA 1380GGTGTTCGGC GCTGACGCAA TGCGCGGCCC GCGCGGCCTG AAAGCGGTTG GCCCGTATGT 1440GGTCACCAAA GCGATGGCAT CCGGCGTTTC TGCCTGCCTC GCCACCCCGT TTAAAATTCA 1500TGGCGTTAAC TACTCCATCA GCTCCGCGTG TGCGACTTCC GCACACTGTA TCGGTAACGC 1560AGTAGAGCAG ATCCAACTGG GCAAACAGGA CATCGTGTTT GCTGGCGGCG GCGAAGAGCT 1620GTGCTGGGAA ATGGCTTGCG AATTCGACGC AATGGGTGCG CTGTCTACTA AATACAACGA 1680CACCCCGGAA AAAGCCTCCC GTACTTACGA CGCTCACCGT GACGGTTTCG TTATCGCTGG 1740CGGCGGCGGT ATGGTAGTGG TTGAAGAGCT GGAACACGCG CTGGCGCGTG GTGCTCACAT 1800CTATGCTGAA ATCGTTGGCT ACGGCGCAAC CTCTGATGGT GCAGACATGG TTGCTCCGTC 1860TGGCGAAGGC GCAGTACGCT GCATGAAGAT GGCGATGCAT GGCGTTGATA CCCCAATCGA 1920TTACCTGAAC TCCCACGGTA CTTCGACTCC GGTTGGCGAC GTGAAAGAGC TGGCAGCTAT 1980CCGTGAAGTG TTCGGCGATA AGAGCCCGGC GATTTCTGCA ACCAAAGCCA TGACCGGTCA 2040CTCTCTGGGC GCTGCTGGCG TACAGGAAGC TATCTACTCT CTGCTGATGC TGGAACACGG 2100CTTTATCGCC CCGAGCATCA ACATTGAAGA GCTGGACGAG CAGGCTGCGG GTCTGAACAT 2160CGTGACCGAA ACGACCGATC GCGAACTGAC CACCGTTATG TCTAACAGCT TCGGCTTCGG 2220CGGCACCAAC GCCACGCTGG TAATGCGCAA GCTGAAAGAT TAATTCGCAG TAGGTCGGAG 2280TAGACGCGCC AGCCTCGCAT CCGACGTTAC GCGCCAATGC GGCCTCCGGC ACTAACGCAA 2340AAGGGAACCT GATGGTTCCC TTTTTCACAT CATTGACAAT CGCCGCCAGT TCCAGGCAAA 2400CTTCCCGCTT TGTCGATTTC CTTCTGAAAA GACGTACGCG TTAAATCCTG CCAACGCACC 2460GTAACCCTGA AACCAGAGAG ATGAGACGGG GATACTCCTC GCCTTGCGCT GCATTCTGGA 2520GTAATGCATG ACTGCTGTAA GCCAAACCGA AACACGATCT TTCTGCCAAT TTTTCGCTYT 2580TTCCGCATCG CTTTTTGCGG TTTTTCTTCA CCTACATGAC CCGTAGGGTT GCCGTTGCCG 2640GTTATCCCGC TGTTTGTT 2658 21 base pairs nucleic acid single linear 15TAAATTCGAG GTTGGCAGGT T 21 20 base pairs nucleic acid single linear 16AATCGACAAA GCGGGAAGTT 20 129 base pairs nucleic acid single linear cDNA17 CTCTCTGGGC GCTGCTGGCG TACAGGAAGC TATCTACTCT CTGCTGATGC TGGAACACGG 60CTTTATCGCC CCGAGCATCA ACATTGAAGA GCTGGACGAG CAGGCTGCGG GTCTGAACAT 120CGTGACCGA 129

What is claimed is:
 1. A method for obtaining a bacterium havingenhanced viability or enhanced transformation efficiency during storageat low temperatures, said method comprising: a. modifying a bacteriumsuch that the fatty acid content of said bacterium is altered; and b.isolating a modified bacterium having enhanced viability or enhancedtransformation efficiency during storage at low temperatures.
 2. Themethod of claim 1, wherein said modification step comprises geneticallyaltering said bacterium.
 3. The method of claim 1, wherein saidmodification step comprises modifying one or more genes involved inchanging, unsaturated fatty acid content of said bacterium.
 4. Themethod of claim 1, wherein said modification step comprises increasingthe amount of one or more unsaturated fatty acids in said bacterium. 5.The method of claim 3, wherein said modification step comprisesenhancing expression of one or more of said unsaturated fatty acidgenes.
 6. The method of claim 5, wherein said enhanced expressioncomprises increasing copy number of said genies.
 7. The method of claim5, wherein said enhanced expression comprises increasing transcriptionor translation of said genes.
 8. The method of claim 1, wherein saidbacterium is a gram negative bacterium.
 9. The method of claim 8,wherein said bacterium is Escherchia.
 10. The method of claim 9, whereinsaid bacterium is E. coli.
 11. The method of claim 1, wherein said fattyacid is an unsaturated fatty acid selected from the group consisting ofoleic acid, linoleic acid, palmitoleic acid, and cis-vaccenic acid. 12.The method of claim 11, wherein said unsaturated fatty acid is selectedfrom the group consisting of cis-vaccenic acid and palmitoleic acid. 13.The method of claim 1, wherein said modified bacterium has an alteredunsaturated fatty acid content in the bacterial membrane.
 14. The methodof claim 1, wherein said low temperatures range from about −20° C. toabout 4° C.
 15. A method for enhancing viability or enhancingtransformation efficiency of a bacterium during storage at lowtemperatures, said method comprising altering the fatty acid content ofsaid bacterium.
 16. The method of claim 15, wherein said modificationstep comprises genetically altering said bacterium.
 17. The method ofclaim 15, wherein said modification step comprises modifying one or moregenes involved in changing unsaturated fatty acid content of saidbacterium.
 18. The method of claim 15, wherein said modification stepcomprises increasing the amount of one or more unsaturated fatty acidsin said bacterium.
 19. The method of claim 17, wherein said modificationstep comprises enhancing expression of one or more of said unsaturatedfatty acid genes.
 20. The method of claim 19, wherein said enhancedexpression comprises increasing copy number of one or more of saidgenes.
 21. The method of claim 19, wherein said enhanced expressioncomprises increasing transcription or translation of one or more of saidgenes.
 22. The method of claim 15, wherein said bacterium is a gramnegative bacterium.
 23. The method of claim 22, wherein said bacteriumis Escherchia.
 24. The method of claim 23, wherein said bacterium is E.coli.
 25. The method of claim 15, wherein said fatty acid is anunsaturated fatty acid selected from the group consisting of oleic acid,linoleic acid, palmitoleic acid, and cis-vaccenic acid.
 26. The methodof claim 25, wherein said unsaturated fatty acid is selected from thegroup consisting of cis-vaccenic acid and palmitoleic acid.
 27. Themethod of claim 15, wherein said modified bacterium has an alteredunsaturated fatty acid content in the bacterial membrane.
 28. The methodof claim 15, wherein said low temperatures range from about −20° C. toabout 4° C.
 29. A bacterium produced by the method of any one of claim 1or
 15. 30. A storage stable bacterium, wherein said bacterium has analtered fatty acid content.
 31. The bacterium of claim 30, wherein saidbacterium is competent for transformation.
 32. A bacterium havingenhanced viability or enhanced transformation efficiency during storageat low temperatures, wherein said bacterium has an altered fatty acidcontent.
 33. The bacterium of claim 32, wherein said bacterium has anincreased unsaturated fatty acid content.
 34. The bacterium of claim 32,wherein said bacterium has been modified genetically.
 35. The bacteriumof claim 32, wherein said bacterium comprises one or more modified genesinvolved in changing unsaturated fatty acid content of said bacterium.36. The bacterium of claim 33, wherein said increased unsaturated fattyacid content is caused by enhancing expression of one or more genesinvolved in changing unsaturated fatty acid content in said bacterium.37. The bacterium of claim 36, wherein said enhanced expressioncomprises increasing copy number of one or more of said genes.
 38. Thebacterium of claim 36, wherein said enhanced expression comprisesincreasing transcription or translation of one or more of said genes.39. The bacterium of claim 32, wherein said bacterium is a gram negativebacterium.
 40. The bacterium of claim 39, wherein said bacterium isEscherchia.
 41. The bacterium of claim 40, wherein said bacterium is E.coli.
 42. The bacterium of claim 32, wherein said fatty acids areunsaturated fatty acids selected from the group consisting of oleicacid, linoleic acid, palmitoleic acid, and cis-vaccenic acid.
 43. Thebacterium of claim 42, wherein said unsaturated fatty acid is selectedfrom the group consisting of palmitoleic acid and cis-vaccenic acid. 44.The bacterium of claim 32, wherein said bacterium has an alteredunsaturated content in the bacterial membrane.